Cambridge Isotope Laboratories, Inc.
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RESEARCH PRODUCTS

Stable Isotopes for
Biomolecular NMR

Cambridge Isotope Laboratories, Inc.
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| Stable Isotopes for Biomolecular NMR

WelcomeOver the years, Cambridge Isotope Laboratories, Inc (CIL) has grown to be the world’s leader in production and distribution of stable isotopelabeled compounds for biological NMR applications. CIL has partnered with the scientific community in the last decade to enable significant
developments in isotopic labeling of proteins and nucleic acids for magnetic resonance applications by commercializing novel compounds
and reagents.
CIL is pleased to present this catalog for biomolecular NMR. Here you will find the latest in isotope-labeled products to support yourresearch, along with contributions written by some of the world’s most preeminent NMR spectroscopists. We are particularly pleased topresent pieces written by Gerhard Wagner, Lewis Kay, Masatsune Kainosho, Leonid Brown and Vladimir Ladizhansky, Hanudatta Atreya,
Tim Cross, and Robert Griffin.
Also included in this catalog is a summation of CIL application notes relevant to magnetic resonance studies of biological moleculesand systems. These application notes help illustrate the usefulness of our products for both solution and solid-state NMR applications.
We would like to thank the many researchers who have written application notes. Also included is our most recent application note written
by Rui Huang and Lewis Kay titled “Stereospecific Leu / Val Methyl Labeling: An Important Technology for NMR Studies of High-MolecularWeight Complexes” (Application Note 48).
As new technologies and applications to study biopolymers using NMR advance, CIL continues to maintain a leadership role in developing
new products for the benefit of the entire biomolecular NMR community. CIL welcomes suggestions from our customers on additional
products that would be beneficial to their research. It has been through our partnerships and close relationships with our customers
over the past 35 years that we have been able to significantly expand our product offering in order to assist the biomolecular NMR
community in the advancement of their studies utilizing stable isotope-labeled compounds as a tool for NMR discovery.
All of us at CIL appreciate the collaborative relationships we have with our customers. We look forward to continuing to work withyou to expand our product offerings in ways that will best fulfill your needs.

Kevin Millis, PhD
Senior Scientist
Applications Development

You can find all of the information presented in this catalog on isotope.com. Visitors to our website can immediately accessupdated product information and documentation, such as a certificate of analysis (CoA) and safety data sheets (SDS) and
can also find real-time product availability. Visit isotope.com to learn more.

Cambridge Isotope Laboratories (CIL) is the world leader in the separation and manufacture ofstable isotopes and stable isotope-labeled compounds. For over 35 years, CIL has remained thepremier supplier of stable isotopes for NMR and MRS / MRI research applications. These products
include a diverse line of RNA / DNA products, minimal media reagents (carbohydrates, ammonium
salts), cell-free expression reagents and kits, free and protected amino acids, as well as cell-growth
media for eukaryotic and prokaryotic cell lines. Additionally, CIL offers a comprehensive line of
deuterated solvents, detergents, and buffers. Our products have been specifically designed andtested with the most discerning NMR spectroscopist and structural biologist in mind. CIL actively
supports the world’s NMR community through meeting sponsorships and customer collaborations.

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| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Table of Contents
Ordering and Contact Information

5

Amino Acids

28

Shipping Information

6

Protected Amino Acids

34

Product Information

6

Sparse Labeling Reagents

37

Mixed Pyruvate Labeling

37
38

Investigator Spotlight

Utility of Stable Isotopes in Structural Biology

7

Minimal Media Reagents

9

Deuterated Detergents and Phospholipids
for Membrane Proteins

Methyl Labeling

10

Investigator Spotlight

The Magic of MAS DNP

Investigator Spotlight

Alanine Probes of Supramolecular Structure
and Dynamics

11

Prochiral Precursors

12

Investigator SpotlightRich E. coli Media

13

Yeast Media and Reagents

16

Investigator Spotlight

O Solid State NMR Spectroscopy:
Coming of Age

40

17

O NMR Reagents

41

Miscellaneous Protein Reagents

42

Protein NMR Standards

44
45
50

17

Pichia pastoris as a Eukaryotic Protein
Isotope-Labeling System

16

Protein Standards from
Nexomics Biosciences, Inc.

Insect Cell Media

18

Nucleic Acids

Mammalian Cell Media

19

Cell-Free Production of Stable
Isotope-Labeled Proteins20

Cell-Free Protein Expression

21

Selective Amino Acid-Type Labeling

26

Investigator Spotlight

Selective Isotope-Labeling Methods
for Protein Structural Studies

4

39

26

tel: +1.978.749.8000 | 1.800.322.1174 (USA) | fax: +1.978.749.2768

Ribonucleic Acids

51

Fluorine-Labeled Nucleotide
5’-Triphosphates

51

Deoxyribonucleic Acids

53

Miscellaneous RNA and DNA Products

55

Deuterated Buffers

56

Solvent Data Chart

57

Application Notes Summaries58

| Stable Isotopes for Biomolecular NMR

isotope.com

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| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Shipping Information

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6

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| Stable Isotopes for Biomolecular NMR

isotope.com

Utility of Stable Isotopes in Structural Biology
Gerhard Wagner, PhD
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts USA

The use of stable isotopes has empowered NMR to becomea major player in mechanistic structural biology and drug discovery.
Over the past quarter century introduction of new isotope-labelingprocedures has led to several quantum leaps in the advancementof NMR spectroscopy. By introducing new NMR active nuclei, or
replacing protons with deuterons, isotope labeling is used to obtainnew correlations and to reduce complexity of spectra. Thus, it hastremendously facilitated obtaining specific structural and dynamic
information. New isotope-labeling procedures have stimulated thedevelopment of a plethora of new NMR experiments. Today,labeling is an integral element of current state-of-the-art NMR
technology and can be expected to play a major role in future
develop­ments. While new labeling methods typically originate
from academic laboratories, it is usually not economical to cultivate
procedures for every isotope-producing procedure in all interested
laboratories with student and post-doc members. This is best
pursued by companies, such as CIL, that can provide sustainablelong-term isotope commodities to the scientific community andare an invaluable service for the NMR community.

Today, labeling is an integral element of current stateof-the-art NMR technology and can be expected toplay a major role in future develop­ments.
Until the early 1970s NMR spectroscopy was a method primarily
used by chemists for characterizing small molecules, checking
chemical structures, and purity of reaction products. Studies ofproteins and nucleic acids became feasible with the arrival ofhigh-resolution superconducting magnets at higher field andnew consoles for pulsed NMR, development of double-resonanceexperiments, intelligent use of the nuclear Overhauser effect, andeventually the introduction of 2D NMR. This allowed collection ofnumerous structural parameters, and with the development of newcomputer programs, the first solution structures were determinedwith NMR in the early to mid 1980s. However, the method waslimited to proteins up to 10 kDa or 15 kDa, and the process wasvery time consuming.
Fortunately, techniques were developed for producing isotopelabeled proteins in bacterial expression systems.1,2 The availabilityof 15N labeled proteins enabled a first quantum leap. Threedimensional 15N-dispersed NOESY experiments were developed
leading to a tremendous gain in signal dispersion and spectral
simplification. Larger structures could be obtained in shorter time
and with higher precision. Access to routine production of 15N
and 13C-labeled proteins has also opened an entirely new field ofprotein dynamics, which is thriving ever since. It has provided andis yielding new insights into how proteins fluctuate and performfunctions, such as in enzymes.

Investigator
Spotlight

The possibility to produce 15N / 13C labeled proteins has madepossible the development of triple-resonance experiments forconformation-independent sequential assignments. Theseexperiments caused a second quantum leap in protein NMR andmade assignments more reliable and very efficient. Triple resonanceexperiments form today the basis of all assignments of proteins.
Another quantum leap was the introduction of deuteration by overexpressing proteins in 2H2O, which reduces dipolar broadening ofresonances by proton dilution.3 Spectroscopy is essentially donewith the amide protons that can be reintroduced by dissolving
deuterated proteins in H2O for measurements. The reduction in linewidths of amide proteins was dramatic and excited everyone in thefield when this was introduced. Deuteration is a key requirement
for the performance of TROSY (Transverse Relaxation Optimized
Spectroscopy) experiments, which brought another performanceleap and is today used for nearly all NMR studies of larger proteins.
With deuterated proteins, backbone assignments are rather
routine even for rather large proteins. The challenge is to havea sufficiently large number of probes for measuring distanceconstraints. Thus, introduction of selectively protonated groups
into deuterated proteins has become very important. Most
prominent is the introduction of protonated ILV methyls, whichuses 13C a-ketoisovalerate and a-ketobutyrate as precursors
for labeling. There is a wide range of applications to introduce
protonated groups into deuterated proteins beyond ILV labeling.
Most sophisticated is the SAIL (stereo-arrayed isotope labeling)
approach, which minimizes dipolar broadening while maintaining
a large number of protons for distance measurements.
In our laboratory, the use of ILV labeling in a deuterated
background, spiked with few protonated residues, hasallowed solving several large structures, where the prime sourceof distance constraints came from methyl-methyl and methylaromatic contacts. The clear separation of the methyl signals fromthe amides as the only protons made possible recording multiple
high-resolution 4D NOESYs in a time-shared manner within areasonable time.4 Figure 1 shows the structure of the enterobactin
synthase 37 kDa EntF T-TE di-domain where most distance restraints
were obtained from such isotope-labeled samples.5
Recent developments have explored additional possibilities for theuse of isotopes to enhance NMR spectroscopy. This includes theadvent of new cryogenic probes that are optimized for 13C and 15N
direct detection. First used for direct detection of carbonyl carbons,
direct detection seemed primarily suited for paramagnetic proteins
where carbons could be observed closer to the paramagnetic center
Continued ➤cilsales@isotope.com | intlsales@isotope.com | isotope.com 7

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Utility of Stable Isotopes in Structural Biology (continued)
Compared to proteins, the use of isotopes has been slower in NMR
of nucleic acids. Due to a lower proton density, dipolar broadening
is less of a problem in nucleic acids. Also connectivity throughphosphorus is inefficient because of the large chemical shiftanisotropy. Thus, assignments are mainly made with NOE
correlations. However, novel labeling methods 9 and intelligent
use of inserting deuterated nucleotides has made tremendous
impact for structural studies of large nucleic acids.10
Considering the great impact isotope labeling had in thedevelopment of NMR in the past, it can be anticipated that
it will play an equal or even greater role in the future.

Acknowledgement
Large amounts of stable isotopes were purchased with support
from the NIH grant 047467.

Figure 1. Structure of the 37 kDa EntF T-TE di-domain.5

than protons. However, it became obvious that direct 13C detection
can be quite powerful for any protein when using alternate 13C-12C
labeling, which can be obtained when using [2-13C] or [1,3-13C2]
glycerol as a carbon source as originally described by LeMaster.6
This eliminates most one-bond carbon-carbon couplings. Whenthe protein is deuterated in addition, carbon resonances become
extremely sharp and new long-range correlations can be observed
through backbone and side chain nuclei.7,8 This is particularlyinteresting for assigning prolines, which are often found adjacentto phosphorylation sites in regulatory domains of proteins and are
difficult to characterize with traditional NMR methods. It is likelythat future developments of isotope labeling will enhance thisapproach.
Currently, the biggest hurdle in isotope-labeling proteins for NMR
studies is the lack of efficient methods for producing isotopelabeled proteins in eukaryotic or mammalian expression systems.
Many mammalian proteins of interest are misfolded when
expressed in bacterial systems due to the lack of folding machinerythat is present in eukaryotic cells, such as those for forming
disulfides. Production in insect cells, yeast, or CHO (Chinese
hampster ovary) cells is possible using special growth media,
but the process is still far from being efficient for wide use.

8

tel: +1.978.749.8000 | 1.800.322.1174 (USA) | fax: +1.978.749.2768

References
1.	LeMaster, D.M.; Richards, F.M. 1985. Proton-nitrogen-15 heteronuclear NMR
studies of Escherichia coli thioredoxin in samples isotopically labeled by residue
type. Biochem, 24, 7263-7268.
2.	McIntosh, L.P.; Dahlquist, F.W.; Redfield, A.G. 1987. Proton NMR and NOE
structural and dynamic studies of larger proteins and nucleic acids aided by isotope
labels: T4 lysozyme. J Biomol Struct Dyn, 5, 21-34.
3.	LeMaster, D.M. 1989. Improved large scale culture of Methylophilus
methylotrophus for 13C / 15N labeling and random fractional deuteration ofribonucleotides. Methods Enzymol, 177, 23-43.
4.	Frueh, D.P.; Vosburg, D.A.; Walsh, C.T.;et al. 2006. Nonuniformly sampled doubleTROSY hNcaNH experiments for NMR sequential assignments of large proteins.
J Biomol NMR, 34, 31-40.
5.	Frueh, D.P.; Arthanari, H.; Koglin, A.; et al. 2008. Dynamic thiolation-thioesterase
structure of a nonribosomal peptide synthetase. Nature, 454, 903-906.
6.	LeMaster, D.M. 1996. Protein structure refinement using 13Cα chemical shifttensors. J Am Chem Soc, 118, 9255-9264.
7.	Takeuchi, K.; Ng, E.; Malia, T.J.; et al. 2007. 1-13C amino acid selective labeling in a
2 15
H N background for NMR studies of large proteins. J Biomol NMR, 38 (1), 89-98.
8.	Takeuchi, K.; Frueh, D.P.; Sun, Z.Y.; et al. 2010. HNCA-TOCSY-CANH experimentswith alternate 13C-12C labeling: a set of 3D experiment with unique suprasequential
information for mainchain resonance assignment. J Biomol NMR, 47, 55-63.
9.	Thakur, C.; Sama, J.; Jackson, M.; et al. 2010. Selective 13C labeling of nucleotides
for large RNA NMR spectroscopy using an E. coli strain disabled in the TCA cycle.
J Biomol NMR, 48, 179-192.
10.	Lu, K.; Miyazaki, Y.; Summers M.F. 2010. A topical issue: production and labelingof biological macromolecules for NMR investigations J Biomol NMR, 46, 113-125.

| Stable Isotopes for Biomolecular NMR

isotope.com

Minimal Media ReagentsThe E. coli expression system using minimal media is the mostcommon means of overexpressing isotope-enriched recombinant
protein for NMR investigations, primarily because the use ofminimal media is the most economical way to grow isotope-labeledbacteria. Minimal media is a mixture M9 salts, glucose (as the solecarbon source), and ammonium chloride or ammonium sulfate(as the sole nitrogen source) that is formulated in either H2O
or D2O. Often, depending on the E. coli strain used, additional
reagents, such as cofactors, are added to the media to enhance
growth.
For proteins greater than ~25 kDa in size, deuteration is generally
required to simplify spectra and reduce the deleterious effects ofline-broadening associated with 1H dipolar coupling. Therefore,
minimal media used to express such proteins must be formulatedusing D2O. For the investigations of large proteins or protein
complexes, fully deuterated glucose as the carbon source isrequired. In particular, it is advantageous to use deuterated
glucose in conjunction with the ILV selective methyl-labelingstrategies that require a highly deuterated protein. The use of
protonated 13C6 glucose as the carbon source may still be
acceptable in some situations, because the level of incorporationof deuterium from the solvent into the expressed proteins is still
significant (e.g., ~70-80%). 13C6 glucose in minimal media
formulated in D2O may also be used to label all protein methylgroups,1 although CHD2-detected variants must be used to remove
signals from the CH3 and CH2D isotopomers in 1H-13C correlation
spectra.
To aid in handling multiple simultaneous growths, auto-induction
methods have been developed which utilize minimal media
containing glucose, glycerol, and lactose. Auto-induction methods
utilize the preference of E. coli containing lac operon-derived
expression systems (e.g., pET, pGEM, and pQE) to selectively use
different carbon sources during diauxic growth. Hence, autoinduction methods will lead to growth on alternative carbon
sources such as glycerol or lactose after exhaustion of glucose in
the growth medium. The basis for auto-induction and adaptation
to 13C,15N was reported by Studier 2 and further optimized forhigh-throughput production by Tyler, et al.3

The table below shows which labeled reagents to use in minimal
media to obtain the highest level of incorporation of the desiredlabeling pattern.
Table: Minimal media reagents used to express single-, double- andtriple-labeled protein.
Desired Labeling
Pattern

N
C
D
13
C,15N
15
N, D
13
C, D
13
C,15N, D
15
13

N NH4Cl or(15NH4)2SO4

D-Glucose(13C6)

D-Glucose(13C6; D7)

D-Glucose
(D7)

D2O

+
+
+
+

+
+
-

+
+

+
+
-

+
+
+
+

15

Minimal Media Reagents for Uniform Labeling
Catalog No.
CLM-1396
DLM-2062
CDLM-3813
CLM-1510
DLM-558
NLM-467
NLM-713
DLM-4-99
DLM-4-99.8
DLM-4

Description
D-Glucose (13C6, 99%)
D-Glucose (1,2,3,4,5,6,6-D7, 97-98%)
D-Glucose (13C6, 99%; 1,2,3,4,5,6,6-D7, 97-98%)
Glycerol (13C3, 99%)
Glycerol (D8, 99%)
Ammonium chloride (15N, 99%)
Ammonium sulfate (15N2, 99%)
Deuterium oxide (D, 99%)
Deuterium oxide (D, 99.8%)
Deuterium oxide (D, 99.9%)

References
1.	Otten, R.; Chu, B.; Krewulak, K.D.; et al. 2010. Comprehensive and cost-effective
NMR spectroscopy of methyl groups in large proteins. J Am Chem Soc, 132,2952-2960.
2.

S tudier, F. W. 2005. Protein production by auto-induction in high-density shaking
cultures. Protein Expr Purif, 41(1), 207-34.

3.	Tyler, R.C.; Sreenath, H.K.; Singh, S.; et al. 2005. Auto-induction medium for the
production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening andstructure determination. Protein Expr Purif, 40, 268-278.

Although minimal media is the most economical to label E. coli,
the resulting growth rate and expression levels are sometimes notsuitable to conduct the desired studies. An alternate method used
to enhance protein yield and growth characteristics is to spike lowlevels of suitably labeled rich media into minimal media prior toinduction. Please see CIL Application Note 12 at isotope.com for
additional information.

cilsales@isotope.com | intlsales@isotope.com | isotope.com 9

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Methyl LabelingProtonated 13C methyl groups in deuterated proteins are excellent
probes for structure and dynamics, and are especially crucial to
study high-molecular weight proteins and protein complexes.
The popular ILV labeling strategy utilizes labeled biosynthetic
precursors to produce protein containing either uniformly or
selectively labeled isoleucine, leucine, and valine. α-Ketoisobutyrate
is the precursor to Ile and may be used to label the δ1 methyl in Ile.
α-Ketoisovalerate is the precursor to Leu and Val and is used tolabel one or both methyl groups in these amino acids. Please see
CIL application notes 16 and 25 at isotope.com for more
information regarding this application.
CIL is pleased to offer a large selection of α-ketobutyric andα-ketoisovaleric acids, along with pyruvate, for use in selective

methyl and side-chain labeling. Uniform 13C-labeled forms of the
precursors are used in conjunction with 13C6 glucose to produce
uniform 13C-labeled Ile and Leu.
CIL also offers kits of conveniently packaged reagents for labelingproteins with isoleucine, leucine, valine, and alanine (i.e., “ILVA”
labeling) or alanine only. These kits are for use with 1 L amountsof deuterated minimal media.1,2 Please see CIL Application Note
25 at isotope.com for more details regarding ILVA and alaninelabeling. Methionine (2,3,3,4,4-D5, methyl-13CH3) and threonine(4-13C; 2,3-D2) are also available for use with deuterated minimal
media to provide new methyl probes in addition to Leu, Ile, Val,
and Ala.

a-Keto Acids
Catalog No.
CLM-6820
CLM-6164
DLM-9168
CDLM-4611
CDLM-7318
CDLM-7353
CLM-6821
CLM-4418
DLM-4646
CDLM-4418
CDLM-7317
CDLM-7354
CDLM-10647
CDLM-8446
CDLM-8100
CLM-1575
CLM-2440

Description
a-Ketobutyric acid, sodium salt (methyl-13C, 99%)
a-Ketobutyric acid, sodium salt (13C4, 98%)
a-Ketobutyric acid, sodium salt (3,3-D2, 98%)
a-Ketobutyric acid, sodium salt (13C4, 98%; 3,3-D2, 98%)
a-Ketobutyric acid, sodium salt (methyl-13C, 99%; 3,3-D2, 98%)
a-Ketobutyric acid, sodium salt (4-13C, 99%; 3,3,4,4-D4, 98%)
a-Ketoisovaleric acid, sodium salt (dimethyl-13C2, 99%)
a-Ketoisovaleric acid, sodium salt (13C5, 98%)
a-Ketoisovaleric acid, sodium salt (D7, 98%)
a-Ketoisovaleric acid, sodium salt (13C5, 98%; 3-D, 98%)
a-Ketoisovaleric acid, sodium salt (3-methyl-13C, 99%; 3,4,4,4-D4, 98%)
a-Ketoisovaleric acid, sodium salt (3-methyl-13C, 99%; 3-methyl-D2, 3,4,4,4-D4, 98%)
a-Ketoisovaleric acid, sodium salt (dimethyl-13C2, 99%; 3-D, 98%)
a-Ketoisovaleric acid, sodium salt (dimethyl-13C2, 98%; 3-methyl-D2, 4,4-D2, 98%)
a-Ketoisovaleric acid, sodium salt (1,2,3,4-13C4, 99%; 3,4’,4’,4’-D4, 97-98%)
Sodium pyruvate (3-13C, 99%)
Sodium pyruvate (13C3, 99%)

Unit Size
0.5 g
0.5 g
Please inquire
0.1 g, 0.25 g
0.5 g, 1 g
0.25 g0.5 g0.25 g, 1 g
Please inquire
0.25 g
0.5 g, 1 g
0.25 g
0.1 g, 0.5 g
0.25 g0.25 g
0.1 g, 0.5 g, 1 g
0.5 g, 1 g

Catalog No.
CDLM-8649

Description
L-Alanine (3-13C, 99%; 2-D, 96%)

CLM-206
CDLM-8885

L-Methionine (methyl-13C, 99%)
L-Methionine (2,3,3,4,4-D5, 98%; methyl-13CH3, 99%)

CDLM-9307
CLM-9217

L-Threonine (4-13C, 97%; 2,3-D2, 96-98%)
L-Valine (dimethyl-13C2, 99%)

Unit Size
1g
1g
0.5 g, 1 g
0.1 g, 0.5 g
0.25 g, 1 g

Amino Acids

CDLM-8805-KIT – in vivo Alanine Methyl-Labeling Kit
Catalog No.
DLM-584
DLM-4646
DLM-141
CDLM-8649

Kit Components (four vials)
Succinic acid (D4, 98%)
a-Ketoisovaleric acid, sodium salt (D7, 98%)
L-Isoleucine (D10, 98%)
L-Alanine (3-13C, 99%; 2-D, 96%)

Unit Size
2.5 g
0.2 g
0.06 g
0.8 g

The materials in this kit are to be used in conjunction with 1 L of deuterated minimal media.1,2 1 L of minimal media that is suitable for use with this producttypically contains 2 g of D-glucose-D7, 1 g of ammonium salt, and 11.3 g of M9 salts in D2O.

10

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| Stable Isotopes for Biomolecular NMR

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CDLM-8806-KIT – in vivo ILVA Methyl-Labeling Kit
Catalog No.
DLM-584
CDLM-7317
CDLM-7318
CDLM-8649

Kit Components (four vials)
Succinic acid (D4, 98%)
a-Ketoisovaleric acid, sodium salt (methyl-13C, 99%; 3,4,4,4-D4, 98%)
a-Ketobutyric acid, sodium salt (methyl-13C, 99%; 3,3-D2, 98%)
L-Alanine (3-13C, 99%; 2-D, 96%)

Unit Size
2.5 g
0.12 g0.06 g
0.8 g

The materials in this kit are to be used in conjunction with 1 L of deuterated minimal media.1 1 L of minimal media that is suitable for use with this producttypically contains 2 g of D-glucose-D7, 1 g of ammonium salt, and 11.3 g of M9 salts in D2O.
References
1. A
 yala, I.; Sounier, R.; Use, N.; et al. 2009. An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein. J Biomol NMR, 43(2),111-119.
2.	Isaacson, R.L.; Simpson, P.J.; Liu, M.; et al. 2007. A new labeling method for methyl transverse relaxation-optimized spectroscopy NMR spectra of alanine residues.
J Am Chem Soc, 129(50), 15428-15429.

Alanine Probes of Supramolecular Structure and Dynamics
Lewis Kay, PhD
Department of Chemistry, University of Toronto, Ontario, Canada
The development of new protein labeling strategies, along with
optimized experiments that exploit the label, have significantly
impacted on the types of biochemical problems that can now be
addressed by solution NMR spectroscopy. One popular strategy instudies of high molecular weight proteins involves the use of a pairof a-keto acids, a-ketobutyrate and a-ketoisovalerate, which serveas the biosynthetic precursors for the production of Ile and Leu/Val,
respectively.1 Addition of these precursors to highly deuterated
protein expression media produces 2H, Ile, Leu, Val- methyl-labeledproteins. These precursors are available with different methyl
isotopomers (13CH3, 13CH2D, 13CHD2) so that a large variety oflabeled proteins can be produced2 and a correspondingly large
number of experiments can be performed using 1H, 13C or 2H nuclei.

The development of new protein labeling strategies,
along with optimized experiments that exploit thelabel, have significantly impacted on the types ofbiochemical problems that can now be addressedby solution NMR spectroscopy.
Ile, Leu, Val methyl groups are powerful probes of side-chain
structure and dynamics, and their utility has been described in asignificant number of papers.2 Ala methyls, on the other hand,report on properties of the backbone and hence provide importantcomplementary information. A number of recent publications
outline approaches for the production of highly deuterated,
Ala-[13CH3] labeled proteins.3,4 Ala labeling is challenging since
this residue is produced directly as a result of transaminationof pyruvate, which is also a precursor in the production of the
branched-chain amino acids. Transamination is reversible so evenif free methyl-labeled Ala is provided to the media, scrambling will

Investigator
Spotlight

occur with label incorporated at a variety of potentially undesired
locations. Recently Boisbouvier and coworkers have developed aprocedure to generate methyl labeling at Ala side chains withminimal (<1%) scrambling.3 This was achieved by adding
2-[2H],3-[13C]-Ala (800 mg / L) as well as precursors for otherpathways in which the scramb led amino acids are produced.
The ability to produce highly deuterated, Ala-[13CH3] labeledproteins further increases the number of methyl probes available
for studies of very high molecular weight systems.5 A number
of applications involving Ala methyl probes can be envisioned,
including measurement of backbone dynamics through relaxation
studies, probing structure via residual dipolar couplings, methylmethyl NOEs (nuclear Overhauser effect) or PREs (paramagnetic
relaxation enhancement) and studies of molecular interactions.

References
1.	Goto, N.K.; Gardner, K.H.; Mueller, G.A.; et al. 1999. A robust and cost-effective
method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-,
2
H-labeled proteins. J Biomol NMR, 13(4), 369-374.
2.	Tugarinov, V.; Kay, L.E. 2005. Methyl groups as probes of structure and dynamics
in NMR studies of high-molecular-weight proteins. ChemBioChem, 6(9), 1567-1577.
3.	Ayala, I.; Sounier, R.; Use, N.; et al. 2009. An efficient protocol for the complete
incorporation of methyl-protonated alanine in perdeuterated protein. J Biomol NMR,
43(2), 111-119.
4.	Isaacson, R.L.; Simpson, P.J.; Liu, M.; et al. 2007. A new labeling method formethyl transverse relaxation-optimized spectroscopy NMR spectra of alanine
residues. J Am Chem Soc, 129(50), 15428-15429.
5.	Amero, C.; Schanda, P.; Dura, M.A.; et al. 2009. Fast two-dimensional NMR
spectroscopy of high molecular weight protein assemblies. J Am Chem Soc,
131(10), 3448-3449.

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| Stable Isotopes for Biomolecular NMR

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Prochiral Precursors
The use of methyl-labeled acetolactate as an in vivo prochiralprecursor for the biosynthesis of proR- or proS-13CH3 valine
and leucine in E. coli cells has been developed by Jerome
Boisbouvier and colleagues.1 Using acetolactate precursors
instead of a-ketoisovaleric acid as a metabolic precursor reduces
the number of labeled methyl cross-peaks by a factor of two.
The resulting simplification of the methyl region of the acquired
spectra makes the study of larger proteins possible and/or moremanageable. In addition, the signal-to-noise ratios for the methyl
cross peaks when using a prochiral precursor are twice as great
when compared to using a-ketoisovaleric acid.

Catalog No.
CDLM-10508-0.25
CDLM-10508-1
CLM-9935-0.25
CLM-9935-1

CIL offers an alternative to acetolactate as a prochiral precursor.
These precursors come as ethyl esters and require the user toperform a hydrolysis step prior to use. Instructions on how
to perform the hydrolysis step are included with each order.

Description
Ethyl 2-hydroxy-2-methyl-3-oxobutanoate (4-13C, 99%; 2-methyl-D3, 98%) (proR)
Ethyl 2-hydroxy-2-methyl-3-oxobutanoate (4-13C, 99%; 2-methyl-D3, 98%) (proR)
Ethyl 2-hydroxy-2-methyl-3-oxobutanoate (methyl-13C, 99%) (proS)
Ethyl 2-hydroxy-2-methyl-3-oxobutanoate (methyl-13C, 99%) (proS)

Package Size
0.25 g1g
0.25 g
1g

Media Volume
1L
4L
1L
4L

Production of Pro-R and Pro-S 13CH3 Labeled Leucine and Valine Using CIL Precursors

Catalog No.

CIL Precursor

in vivo Precursor

Amino Acids

CDLM-10508

hydrolysis
H2O

E. coli[13CH3] pro-R Leu/ Val

CLM-9935

hydrolysis
D 2O

E. coli[13CH3] pro-S Leu/ Val

Reference
1.	Gans, P.; Hamelin, O.; Sounier, R.; et al. 2010. Stereospecific isotopic labeling of methyl groups for NMR spectroscopic studies of high molecular weight proteins. Angew Chem Int
Ed Engl, 49(11), 1958-1962.

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| Stable Isotopes for Biomolecular NMR

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Rich E. coli Media
Although growth in minimal (M9) media is economical, there is no
substitute for the enhanced growth rates and increased levels ofprotein expression that may be gained by the use of a rich medium.
Rich bacterial media are complex formulations that are usuallyderived from algal hydroylsates and contain all the necessary
nutrients to promote excellent growth. CIL offers a number of richmedia used in labeled protein expression using bacterial systems.

BioExpress® 1000
BioExpress 1000 is CIL’s all-time classic rich bacterial cell
growth medium. BioExpress 1000 provides excellent growthand expression characteristics for a number of different bacterial
systems. BioExpress 1000 contains nearly the same level of aminoacids as LB medium. Glucose levels range from 0.1-0.5 g/ L,
depending on the batch. BioExpress 1000 media is preparedby adding sterile cell culture-grade water and mixing. Please notethat D2O is required for reconstitution for products containing
deuterium. BioExpress 1000 is supplied as a 100 mL sterile liquid
10× concentrate, and reconstitutes to 1 L with no final pH
adjustment required; 10 mL sample sizes are also available.
The 10 mL sample size reconstitutes to make 100 mL of
media with no final pH adjustment required. A small amount ofsolid material may be present after dilution of the BioExpress 1000with water. This material should be removed by using the supplied
0.2 µm filter prior to use.
Catalog No.
CGM-1000-C
CGM-1000-D
CGM-1000-N
CGM-1000-CD
CGM-1000-CN
CGM-1000-DN
CGM-1000-CDN
CGM-1000-U

Growth Curves E. coli (BL21, pGFP)

Description
BioExpress 1000 (13C, 98%) 10× concentrate
BioExpress 1000 (D, 98%) 10× concentrate
BioExpress 1000 (15N, 98%) 10× concentrate
BioExpress 1000 (13C, 98%; D, 98%)
10× concentrate
BioExpress 1000 (13C, 98%; 15N, 98%)
10× concentrate
BioExpress 1000 (D, 98%; 15N, 98%)
10× concentrate
BioExpress 1000 (13C, 98%; 15N, 98%;
D, 98%) 10× concentrate
BioExpress 1000 (unlabeled) 10× concentrate

Growth Curves E. coli (M15, pTMK)For pointers on how to maximize protein yield using CIL’s BioExpress
1000 media, please see CIL Application Note 15 at isotope.com.
To learn how spiking BioExpress 1000 media into minimal media
provides a low-cost means to enhance the performance of minimal
media, please see CIL Application Note 12 at isotope.com.
BioExpress® is a registered trademark of Cambridge Isotope Laboratories, Inc.

❛❛In our hands, CIL’s BioExpress 1000 worked like a charm.
The cell growth rate and protein expression level essentiallymatched the results obtained with Luria broth, and the
N labeling efficiency was excellent.❜❜

15

Tero Pihlajamaa, PhD
Institute of Biotechnology
University of Helsinki, Finlandcilsales@isotope.com | intlsales@isotope.com | isotope.com 13

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Rich E. coli Media
Celtone® and Celtone® Plus Powder

Celtone® Complete

Celtone powder is CIL’s most flexible nutrient-rich media.
The advantage of Celtone powder is that researchers canformulate a custom medium based on their specific researchneeds. Depending on cell line and desired performance, thispowdered media can be used at concentrations ranging from
1 g to 10 g per liter. Truly exceptional performance has beenachieved using 10 g of Celtone powder in 1 L of medium
containing M9 salts, 2-3 g glucose and 1 g of ammonium
chloride (see graph on page 13). Because it is a powder, thisproduct has the longest shelf life of any fully rich bacterialcell growth medium.

Celtone Complete yields a growth rate comparable to LB media,
allowing for inoculation and induction within one working day.
Glucose levels range from ~0.3 to 0.6 g/L, depending on the batch.
Celtone Complete contains 10 g of Celtone powder per liter of
media; it is ready-to-use and does not require dilution or pH
adjustment. Each lot is tested for sterility, cell growth and protein
expression. Celtone Complete is available in 0.1 L and 1 L sizes.

Celtone powder is available in 0.5 g and 1 g packaged sizes.
Celtone Plus base powder contains uniformly enriched aminoacids and peptides along with a collection of nutrients that
promote robust growth for BL21 cells. Celtone Plus contains
over twice the nutrients of Celtone, providing the highest levelsof protein expression yields of any CIL rich bacterial cell growth
media. Celtone Plus is added to minimal media at concentrations
from 1 to 10 g/L.
Celtone Plus is available in 1 g and 10 g packaged sizes.
Catalog No.
CGM-1030P-C
CGM-1030P-D
CGM-1030P-N
CGM-1030P-CN
CGM-1030P-DN
CGM-1030P-CDN
CGM-1030P-U
CGM-1050P-C
CGM-1050P-D
CGM-1050P-N
CGM-1050P-DN
CGM-1050P-CDN
CGM-1050P-U

Description
Celtone Base Powder (13C, 98%)
Celtone Base Powder (D, 97%)
Celtone Base Powder (15N, 98%)
Celtone Base Powder (13C, 98%; 15N, 98%)
Celtone Base Powder (D, 97%; 15N, 98%)
Celtone Base Powder (13C, 98%; D, 97%; 15N, 98%)
Celtone Base Powder (unlabeled)
Celtone Plus Base Powder (13C, 97-99%)
Celtone Plus Base Powder (D, 97%+)
Celtone Plus Base Powder (15N, 97-99%)
Celtone Plus Base Powder (D, 97-99%; 15N, 97-99%)
Celtone Plus Base Powder(13C, 97-99%; D, 97-99%; 15N, 97-99%)
Celtone Plus Base Powder (unlabeled)

Catalog No.
CGM-1040-C
CGM-1040-D
CGM-1040-N
CGM-1040-CN
CGM-1040-DN
CGM-1040-CDN
CGM-1040-U

Description
Celtone Complete Medium (13C, 98%)
Celtone Complete Medium (D, 97%)
Celtone Complete Medium (15N, 98%)
Celtone Complete Medium (13C, 98%; 15N, 98%)
Celtone Complete Medium (D, 97%; 15N, 98%)
Celtone Complete Medium(13C, 98%; D, 97%; 15N, 98%)
Celtone Complete Medium (unlabeled)

Spectra 9 Media
Spectra 9 Media is not a fully rich medium, however, it represents
a cost-effective medium for E. coli growth and protein expression.
This ready-to-use liquid medium is comprised of labeled salts,labeled carbohydrates (>2 g/L glucose), and contains Celtone
powder at a concentration of 1 g/L.
Catalog No.
CGM-3030-C
CGM-3030-D
CGM-3030-N
CGM-3030-CN
CGM-3030-DN
CGM-3030-CDN
CGM-3030-U

Description
Spectra 9 (13C, 98%)
Spectra 9 (D, 97%)
Spectra 9 (15N, 98%)
Spectra 9 (13C, 98%; 15N, 98%)
Spectra 9 (D, 97%; 15N, 98%)
Spectra 9 (13C, 98%; D, 97%; 15N, 98%)
Spectra 9 (unlabeled)

BioExpress® is a registered trademark of Cambridge Isotope Laboratories, Inc.
Celtone® is a registered trademark of Cambridge Isotope Laboratories, Inc.

❛❛The customer service at Cambridge Isotope Labs is
Note: If deuterium labeling is desired, D2O must be used in media
preparation.
It is normal to have insoluble material present after dissolution.
This material may be removed using filter paper prior to sterile
filtration and will not affect performance of the medium.

unmatched. Our dedicated sales representative responds
promptly and thoroughly with quotes, product information,
shipping information, services, and other information. Theirproducts are of the highest quality, and we continue to trust
their chemicals and service. Any issue has been resolved
quickly and painlessly. For these reasons we have been
dedicated to CIL. ❜❜Victor Beaumont, PhD student
Loria Group
Department of Chemistry
Yale University

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| Stable Isotopes for Biomolecular NMR

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Rich E. coli Media
Recommended Testing Protocol for BioExpress 1000, Celtone Media, and Spectra 9
All necessary substrates for optimal growth and protein expression are provided in the media. Since bacterial cell growth and recombinant
protein expression can be strain specific, this is a general protocol which may need to be modified depending on the specific bacterial
strain used.

Experiment 1: Growth Curve Study

Experiment 2: Expression Test

Day 1

Day 1

1.	Beginning with a freshly grown agar plate, choose an
isolated colony of bacteria and inoculate a 2 mL
culture of Luria broth (LB) (or other standard media)
containing the appropriate antibiotic.
2.	Shake (or rotate in a roller drum) the culture tube
overnight at the appropriate temperature.

1.	Beginning with a freshly grown agar plate, choose
an isolated colony of bacteria and inoculate a 2 mL
culture of LB (or other standard media) containing
the appropriate antibiotic.
2.	Shake (or rotate in a roller drum) the culture tube
overnight at the appropriate temperature using
identical conditions as were used for the growth
curve study.

Day 2
1.	In the early morning, the 2 mL culture should be
dense (OD600 ≥ 4). Inoculate a control media (e.g. LB)
and the labeled media (both containing theappropriate antibiotic) with a 1:100 dilution of theovernight culture.
2.	Shake (or rotate) the cultures at the appropriate
temperature and collect OD600 data at the following
time points (Thours): T0, T2, T3, T4, T5, T6, T7, T8, andapproximately T24 (or overnight).

Day 2
1.	In the early morning check the OD600 of the 2 mL
culture (should be ≥ 4). Inoculate a control media(e.g. LB) and the labeled media (both containing theappropriate antibiotic) with a 1:100 dilution of theovernight culture. Depending on the number of
induction time points to be analyzed, a small shakeflask may be required (instead of a test tube) to growa large-enough expression culture.
2.	For rich growth media, induce expression of therecombinant protein when the culture reaches an
OD600 of 0.6 to 0.8 (I0 ). At the time of induction
remove a 0.5 mL (or 1.0 mL) sample, spin down theculture, and after carefully removing the supernatant,freeze the cell pellet at -20oC.
3.	Remove and process additional culture samples at thefollowing time points (Ihours): I2, I4, I6, I24.
These cell pellets should then be used for SDS PAGE gelanalysis or enzyme activity assays to compare protein
expression and determine the optimum time of induction
for the specific recombinant protein being produced.

Helpful Hints•	In general, good aeration is vital to optimal growth and expression. This requires the use of larger (18-25 mm diameter) culture
tubes or small shake flasks (with baffles, if available) to allow for maximum aeration. Culture volume should be kept to 20-25%
of flask volume.
•	Use freshly streaked agar plates to inoculate the growth and expression cultures.
•	Cells grow approximately half as fast in deuterated media than in protonated media and also require longer induction times to achieve
maximum expression.

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| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Yeast Media and ReagentsThe overexpression of protein in yeast cells represents a powerfulexpression system for the source of properly folded and functional
eukaryotic protein. Please see CIL Application Note 26 at
isotope.com for additional information regarding yeast as
a viable expression system to produce isotope-enriched protein
for NMR investigations.
Silantes® Yeast-OD2 (rich growth media for yeast) are ready-to-use
formulations. The problem of optimizing standard minimal media
containing glucose or methanol as a sole carbon source does notarise when using Yeast-OD2 (available in a 1 L packaged size).
For yeast that grows well in minimal media (e.g. pichia), CIL offers
three different single- and double-labeled carbon sources, as wellas in 15N ammonium salts.
The use of a-ketobutyrate (CDLM-7318) to label the d1-methylof isoleucine has recently been reported in P. pastoris1 which opens
up a new avenue of research related to the expression of isotope
labeling of eukaryotic proteins.
Please see CIL Application Note 26 at isotope.com for additional
information regarding the use of methanol, glucose, andammonium salts for use in yeast.

Yeast Media and Reagents
Catalog No.
CGM-4020-SL-C
CGM-4020-SL-N
CGM-4020-SL-CN
CGM-4020-SL-U-S
NLM-467
NLM-713
CLM-1396
DLM-2062
CDLM-3813
CLM-1510
DLM-558
CDLM-7745
CDLM-7318
CLM-359
CDLM-1035

Description
Yeast-OD2 (13C, 98%)
Yeast-OD2 (15N, 98%)
Yeast-OD2 (13C, 98%; 15N, 98%)
Yeast-OD2 (unlabeled)Ammonium chloride (15N, 99%)
Ammonium sulfate (15N2, 99%)
D-Glucose (13C6, 99%)
D-Glucose (D7, 97-98%)
D-Glucose (13C6, 99%; D7, 97-98%)
Glycerol (13C3, 99%)
Glycerol (D8, 99%)
Glycerol (13C3, 99%; D8, 98%) CP 95%
a-Ketobutyric acid, sodium salt
(methyl-13C, 99%; 3,3-D2, 98%)
Methanol (13C, 99%)
Methanol (13C, 99%; D3, 98%)

Reference
1.	Clark, L.; Zahm, J.A.; Ali, R.; et al. 2015. Methyl labeling and TROSY NMR
spectrosocopy of proteins expressed in the eukaryote Pichia pastoris. J Biomol
NMR, 62(3), 239-245.

Pichia pastoris as a Eukaryotic Protein Isotope-Labeling System
Leonid S. Brown, PhD, Professor, and Vladimir Ladizhansky, PhD, Professor
Department of Physics, University of Guelph, Ontario Canada
The development of new systems for recombinant expressionof isotopically labeled proteins is of significant interest to NMR
spectroscopists. While many prokaryotic proteins can readily
be over-expressed in E. coli and yield functional samples, overexpression of eukaryotic, and, more specifically, medically importantmammalian proteins, often requires a different, nonprokaryotic,
host.1-3 Yeast is an alternative choice of eukaryotic expression
system that has attracted considerable attention in recent years.
Isotopic labeling of proteins in yeast for solution NMR studies began
about 25 years ago.4 Yeast is currently a second most popular
expression system after E. coli, and seems to offer the best of thetwo worlds. These systems have the distinct advantages of low
labeling costs, high expression yields, ease of genetic manipulation,ability to grow on deuterated media, and speeds of expression close
to those in E. coli. At the same time, yeast expression allows native
folding and an array of post-translational modifications, such asproteolytic truncation, formation of disulfide bonds, glycosylation,
phosphorylation, and acylation, typical for eukaryotic cell cultures.
Finally, the ability for secreted expression avoids problems with
toxicity of the expressed proteins.1

16

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Investigator
Spotlight

Yeast is an alternative choice of eukaryotic expression
system that has attracted considerable attention inrecent years.
Among several yeast species used for isotope labeling of proteins,the methylotrophic yeast Pichia pastoris accounts for the lion’s shareof produced structures (about 40 unique proteins).5 Expression in
Pichia pastoris usually results in higher expression yields (especiallyin fermenters) and more native patterns of glycosylation than inother yeast species.6 Thus, Pichia pastoris is the yeast expression
system of choice for today’s NMR studies of eukaryotic proteins.
Another popular choice, Saccharomyces cerevisiae, comes in at a
distant second. So far, it has yielded nine unique protein structures,with no structures published in the last eight years.5 Yet anotheryeast species, Klyuveromyces lactis, was recently evaluated forlabeled protein expression with promising results.1
The uniform 13C/ 15N isotopic labeling in Pichia pastoris is verystraightforward and follows established protocols, both for shaker
incubators and fermenters.6-8 A large variety of strains and vectors

| Stable Isotopes for Biomolecular NMR

isotope.com

Pichia pastoris as a Eukaryotic Protein Isotope-Labeling System (continued)
designed for both secreted and nonsecreted expression is available
from Invitrogen.9 Among the highlights are the availability ofprotease-deficient strains, a number of vectors designed for
efficient protein secretion (or membrane-targeting), protein
tagging, and selection for multicopy integration transformants.
The protocols for the removal of covalently and noncovalently
bound sugars are available as well.10 Pichia pastoris grows wellon minimal media, where 15N can be supplied in the form ofammonium salts, and two different 13C sources are employed in thepreinduction and the postinduction phase. As protein expression is
induced by methanol and is conducted under the strong alcoholoxidase promoter, 13C-labeled methanol is the main carbon sourcein the protein expression phase. Prior to the induction, 13C-labeledglycerol or glucose should be employed.6 Economical protocols tominimize the use of those labeled precursors have been developed.8
Yields of hundreds of milligrams of soluble secreted proteins can beachieved in fermentors at extremely high cell densities. Even flask
cultures can produce tens of milligrams of doubly isotopicallylabeled proteins per liter of culture.7 Important for NMR
applications, a number of deuteration protocols is available,both for efficient backbone deuteration using protonated carbon
sources in D2O, as well as for full deuteration.11,12 Finally, proceduresfor isotopic labeling of selected amino acid types (Cys, Leu, Lys, andMet) have been published, and Pichia strains for labeling aromatic
sidechains have been developed.13,14
A number of interesting structures of proteins expressed in
Pichia pastoris have been solved by solution NMR spectroscopy
in the last two decades, including the Man-6-P receptor diester
recognition domain, cofactor-active fragment of thrombomodulin,collagen-binding domain of discoidin domain receptor 2, and manyothers.5 One can expect that in the near future yeast expression
systems will emerge as a very valuable tool in the NMR studies of
eukaryotic membrane proteins, which are extremely hard to express
functionally in bacterial systems and usually too expensive to
produce in cell cultures. Multiple successful trials of functional
expression of G-protein coupled receptors (GPCRs) and othermembrane proteins in Pichia pastoris on a milligram scale15-17
give us optimism on the future of yeast both for solution andsolid-state NMR spectroscopy.

References
1.	Takahashi, H.; Shimada, I. 2010. Production of isotopically labeled heterologous
proteins in non-E. coli prokaryotic and eukaryotic cells. J Biomol NMR, 46(1), 3-10.

11.	Morgan, W.D.; Kragt, A.; Feeney, J. 2000. Expression of deuterium-isotope-labeledprotein in the yeast pichia pastoris for NMR studies. J Biomol NMR, 17(4), 337-347.

2.	Demain, A.L.; Vaishnav, P. 2009. Production of recombinant proteins by microbes
and higher organisms. Biotechnol Adv, 27(3), 297-306.

12.	Massou, S.; Puech, V.; Talmont, F.; et al 1999. Heterologous expression of a
deuterated membrane-integrated receptor and partial deuteration inmethylotrophic yeasts. J Biomol NMR, 14(3), 231-239.

3.	Lundstrom, K.; Wagner, R.; Reinhart, C.; et al. 2006. Structural genomics onmembrane proteins: comparison of more than 100 GPCRs in 3 expression systems.
J Struct Funct Genomics, 7(2), 77-91.
4.	Laroche, Y.; Storme, V.; De Meutter, J.; et al. 1994. High-level secretion and very
efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the
methylotrophic yeast, Pichia pastoris. Biotechnology (NY), 12(11), 1119-1124.
5. www.rcsb.org
6.	Pickford, A.R.; O‘Leary, J.M. 2004. Isotopic labeling of recombinant proteins fromthe methylotrophic yeast Pichia pastoris. Methods Mol Biol, 278, 17-33.
7.	Wood, M.J.; Komives, E.A. 1999. Production of large quantities of isotopicallylabeled protein in Pichia pastoris by fermentation. J Biomol NMR, 13(2), 149-159.
8.	Rodriguez, E.; Krishna, N.R. 2001. An economical method for N/ C isotopic
labeling of proteins expressed in Pichia pastoris. J Biochem, 130(1), 19-22.
15

13

9. www.invitrogen.com
10.	O’Leary, J.M.; Radcliffe, C.M.; Willis, A.C.; et al. 2004. Identification and removalof O-linked and non-covalently linked sugars from recombinant protein produced
using Pichia pastoris. Protein Expr Purif, 38(2), 217-227.

13.	Chen, C.Y.; Cheng, C.H.; Chen, Y.C.; et al. 2006. Preparation of amino-acid-type
selective isotope labeling of protein expressed in Pichia pastoris. Proteins, 62(1),279-287.
14.	Whittaker, M.M.; Whittaker, J.W. 2005. Construction and characterization of Pichia
pastoris strains for labeling aromatic amino acids in recombinant proteins. Protein
Expr Purif, 41(2), 266-274.
15.	Singh, S.; Hedley, D.; Kara, E.; et al. 2010. A purified C-terminally truncated humanadenosine A(2A) receptor construct is functionally stable and degradation resistant.
Protein Expr Purif, 74(1), 80-87.
16.	Sarramegna, V.; Muller, I.; Mousseau, G.; et al. 2005. Solubilization, purification,and mass spectrometry analysis of the human mu-opioid receptor expressed in
Pichia pastoris. Protein Expr Purif, 43(2), 85-93.
17.	André, N.; Cherouati, N.; Prual, C.; et al. 2006. Enhancing functional productionof G protein-coupled receptors in Pichia pastoris to levels required for structural
studies via a single expression screen. Protein Sci, 15(5), 1115-1126.

cilsales@isotope.com | intlsales@isotope.com | isotope.com 17

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Insect Cell MediaThe baculovirus expression vector system (BEVS), first introducedin the mid-1980s, has grown to become the most versatile andwidely used eukaryotic vector system employed for the expressionof recombinant proteins in cultured insect cells. The BEVS is basedon the infection of insect cells with recombinant baculovirus (BV)
carrying the gene of interest with the subsequent expression of thecorresponding recombinant protein by the insect cells. The mostpopular insect cell lines used in conjunction with the BEVS are Sf9(Spodoptera frugiperda) and High FiveTM (Trichopulsia ni ).

Insect Cell MediaCIL is pleased to offer BioExpress 2000, a rich growth media for
culturing insect cells. The use of BioExpress 2000 in the uniform
15
N and selective amino acid-type labeling of the catalytic domain
of AbI kinase is described in CIL Application Note 14 (see isotope.
com). The use of Experimental Design in the optimization of protein
yield using BioExpress 2000 is exemplified in CIL Application Note
20 (see isotope.com).

Please inquire for custom media.

BioExpress 2000 is packaged as two components: a solid powder(a proprietary blend of inorganic salts, carbohydrates, and labeledamino acids) and a liquid component (fatty acid solution). Selective
amino acid-type labeling is possible with BioExpress 2000 because
the amino acid content is chemically defined.

❛❛My research group develops solid-state NMR methods forprotein structure determination, requiring large quantitiesof 13C,15N-labeled samples produced in-house. We have beenvery pleased with CIL products for isotopically labeled protein
expression, including glucose, glycerol, ammonium chloride,
BioExpress, and specifically labeled amino acids. Using CIL
products, we routinely obtain several milligrams of labeled
membrane proteins per liter of E. coli and tens to hundreds ofmilligrams of water-soluble proteins. We especially appreciate
the competitive pricing, outstanding quality control, friendly
customer service, and timely delivery of a wide range of
products in stock that CIL provides. It’s reassuring to knowthat CIL values its customers and never compromises productquality. ❜❜Chad Rienstra, PhD
Department of Chemistry
University of Illinois

Catalog No.
CGM-2000-N
CGM-2000-N-S
CGM-2000-CN
CGM-2000-U
CGM-2000-U-S
CGM-2000CUSTOM

Description
BioExpress 2000 (15N, 98%)
BioExpress 2000 (15N, 98%) reconstitutes to 200 mL
BioExpress 2000 (13C, 98%; 15N, 98%)
BioExpress 2000 (unlabeled)
BioExpress 2000 (unlabeled) reconstitutes to 200 mL
BioExpress 2000
(labeled amino acids specified by customer)

Protocol for Uniform Isotope Labelingof Proteins with BV-infected Sf9 Cells
1.	Prior to performing isotope labeling of a protein,
optimize culture and BV-infection conditions in
unlabeled medium (e.g. BioExpress 2000-U) for
expression of the protein.
2.	Several 100 mL cultures of Sf9 cells adapted to
growth in serum-free medium SF900 II in 500 mL
Erlenmeyer flasks are cultivated for 3 days at 27°C,
shaken at 90 rpm.
3.	Prepare the uniform isotope-labeling medium (e.g.
BioExpress 2000-CN) according to CIL’s instructions.
It can be stored, filter-sterilized for several months at
4°C in the dark without loss of capacity for protein
expression. Requires warming up to 28°C before use.
4.	When the final cell density of the culture has reached~1.5 × 106 c/mL(~3 days), sterile centrifuge the cells
at 400 G for 20 minutes at 20°C.
5.	Resuspend the pelleted cells in 100 mL portions inlabeled medium (e.g. BioExpress 2000-CN) andtransfer to fresh 500 mL Erlenmeyer flasks.
6.	Add the recombinant baculovirus of a titer of
0.5-2 × 108 pfu/mL to a MOI=1-2, according to
optimized conditions.
7.	The 100 mL cultures of BV-infected Sf9 cells aregrown for 3 days in labeled medium post infection
at 27°C, shaken at 90 rpm.
8.	Harvest the cells expressing the labeled recombinant
protein by centrifugation (400 G, 20 minutes. at20°C); resuspend the pelleted cells in PBS, pH 6.2
with protease inhibitor mix (Complete™, Roche)followed by a second centrifugation in 50 mL plastic
tubes under conditions as above. Store the pelleted
cells at -80°C.
9.	Isolate and purifiy the recombinant protein according
to protocols generated for the unlabeled protein. MS
and NMR analysis are carried out for proteins labeled
in Sf9.
For more information, see CIL Application Note 14 at
isotope.com.

18

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| Stable Isotopes for Biomolecular NMR

isotope.com

Mammalian Cell Media
There is continued interest in obtaining labeled recombinant proteinfrom mammalian cells, because eukaryotic protein expressed in
mammalian cells has the greatest probability of being properly
folded and functional.
CIL offers the only commercially available labeled mammalian media
intended for the production of labeled protein with yields suitablefor NMR studies. Similar growth characteristics have been obtainedusing BioExpress 6000 compared to DMEM (Dulbecco’s modified
Eagle’s medium). The amino acid content in BioExpress 6000 is
chemically defined so many different custom labeling strategies
may be realized. For example, Harald Schwalbe, Karla Werner,
and Judith Klein-Seetharaman at Goethe University have used
BioExpress 6000 to express rhodopsin from HEK293 cells that islabeled at the Gly, Lys, Leu, Gln, Ser, Thr, Val, and Trp residues witheither 15N or 13C/ 15N.1 Please see the presented data regarding theirapplication, as well as a growth curve for unlabeled, 15N-labeled,and 13C/ 15N-labeled BioExpress 6000.
CIL has several different formulations of BioExpress 6000, which
is a powdered medium. The CGM-6500 products do not containsodium pyruvate, allowing users to add a labeled version of sodium
pyruvate to the medium, if desired. The CGM-6750 products
include HEPES to give extra buffering capacity to the culture
medium, which is useful for cells that produce a large amountof lactic acid. The CGM-6800 products do not contain calcium
chloride in order to culture nonadherent cells or multicellularspheroids. Unless otherwise noted, the end product reconstitutes
to 1000 mL of media.

Mammalian Cell Media
Catalog No.
CGM-6000-N
CGM-6000-N-S

Description
BioExpress 6000 (15N, 98%)
BioExpress 6000 (15N, 98%)
reconstitutes to 200 mL
CGM-6000-CN
BioExpress 6000 (13C, 98%; 15N, 98%)
CGM-6000-U-S
BioExpress 6000 (unlabeled)reconstitutes to 200 mL
CGM-6000BioExpress 6000
CUSTOM(labeled amino acids specified by customer)
CGM-6500-N
BioExpress 6000 without sodium pyruvate(15N, 98%)
CGM-6500-CN
BioExpress 6000 without sodium pyruvate(13C, 98%; 15N, 98%)
CGM-6500BioExpress 6000 without sodium pyruvate
CUSTOM(labeled amino acids specified by customer)
CGM-6750-N
BioExpress 6000 with HEPES (15N, 98%)
CGM-6750-CN
BioExpress 6000 with HEPES(13C, 98%; 15N, 98%)
CGM-6750BioExpress 6000 with HEPESCUSTOM(labeled amino acids specified by customer)
CGM-6750-U-0.25 BioExpress 6000 with HEPES (unlabeled)reconstitutes to 200 mL
CGM-6800-N
BioExpress 6000 without CaCl2 (15N, 98%)
CGM-6800-U-S
BioExpress 6000 without CaCl2 (unlabeled)reconstitutes to 200 mL

Please inquire for custom media.

Reference
1.	Werner, K.; Richter, C.; Klein-Seetharaman, J.; et al. 2008. Isotope labeling of
mammalian GPCRs in KEK293 cells and characterization of the C-terminus of bovine
rhodopsin by high resolution liquid NMR spectroscopy. J Biomol NMR, 40(1), 49-53.

This graph shows the number of viable HEK293 cells per mL of culture
for differently labeled CIL mammalian media. Cells are induced on
day three and four and harvested two days later. No differences in celldensities are seen so far. Protein yield is approximately 2.2 mg/L cell
culture in all cases.

C HSQC spectrum of 13C/ 15N-labeled rhodopsin showing the sharper
peaks due to the more flexible region of the spectrum.
13

cilsales@isotope.com | intlsales@isotope.com | isotope.com 19

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Cell-Free Production of Stable Isotope-Labeled Proteins
Masatsune Kainosho, PhD
Tokyo Metropolitan University, Hachioji, Tokyo and Nagoya University, Nagoya, Japan
Nuclear magnetic resonance (NMR) spectroscopy is used forvarious purposes in protein science, such as structural biology
and drug development. During the last two decades, manyof the long-standing methodological difficulties of protein NMR
spectroscopy, such as molecular size and sensitivity limitations,have been successfully addressed. For example, using the SAIL
(stereo-array isotope labeling) method, it is now routinely possible
to determine the high-quality structures of proteins as large as
40-50 kDa, as easily as small proteins.1 These technologicalbreakthroughs emerged by the synergic development ofspectroscopic methodologies and preparative methodsof protein samples optimized for collecting the necessary
NMR parameters in efficient and accurate manners.
A crucial issue was the development of appropriate protein
expression systems, to enable NMR spectroscopists to prepareisotope-labeled protein samples in their own laboratories. It isespecially important when using sophisticated stable isotope-aided
NMR approaches, such as the SAIL method, to choose a methodthat efficiently incorporates the expensive labeled amino acids into
the targeted proteins without serious metabolic scrambling. This iswhere the cell-free protein expression systems have made a major
contribution. The cell-free expression systems utilize the extractsof various living cells, which contain all of the cellular components
relevant for protein synthesis. One can choose the most appropriate
host cells, which can be micro-organism, plant or mammalian cells,
depending on the type of protein one needs to express.

The cell-free expression systems utilize the extractsof various living cells, which contain all of the cellularcomponents relevant for protein synthesis.
The cell-free protein expression system actually has a long
history, dating back to the initial successful trial to express a protein
in vitro using an E. coli cell extract in the 1960s, as described in theliterature.2 In those early days, the protein production stopped soonafter the expression got started, and therefore it was not possible20

tel: +1.978.749.8000 | 1.800.322.1174 (USA) | fax: +1.978.749.2768

Investigator
Spotlight

to prepare the amounts of protein that are required for an NMR
or X-ray structural analysis. During the last few decades, however,
the expression level has been enormously improved by several
key modifications.3 Now, cell-free protein synthesis methods
are commonly used as important alternatives to cellular protein
expression systems, for a wide variety of functional and structural
studies.4,5,6 Although most of the current NMR investigations
employing cell-free protein production utilize the E. coli cell-free
extract, it should be noted that the wheat germ extract seems tobe quite useful to express “difficult” proteins, such as mammalianproteins or large protein complexes.7 In addition to the methods
using cell extracts, there is a completely different approach that
employs a reconstituted protein expression system: the “PUREsystem.” Since the PURE system is exclusively composed of thepurified components necessary for protein synthesis, it will open
various new possibilities that cannot be realized by any othermethods.8
References
1.	Kainosho, M.; Torizawa, T.; Iwashita, Y.; et al. 2006. Optimal isotope labelingfor NMR protein structure determinations. Nature, 440(7), 52–57.
2.	
Kramer, G.; Kudlicki, W.; Hardesty, B. 1999. Cell-free coupled transcriptiontranslation systems from Escherichia coli. Oxford University Press, New York,
pp. 129-165.
3.	
Spirin, A.S.; Baranov, V.I.; Ryabova, L.A.; et al. 1988. A continuous cell-freetranslation system capable of producing polypeptides in high yield. Science,
242(4882), 1162-1164.
4.	Kigawa, T.; Yabuki, T.; Yoshida, Y.; et al. 1999. Cell-free production and stableisotope labeling of milligram quantities of proteins. FEBS Lett, 442(1), 15-19.
5.	
Torizawa, T.; Shimizu, M.; Taoka, M.; et al. 2004. Efficient production of isotopicallylabeled proteins by cell-free synthesis: A practical protocol. J Biomol NMR, 30(3),311–325.
6.	Takeda, M.; Ikeya, T.; Güntert, P.; et al. 2007. Automated structure determinationof proteins with the SAIL-FLYA method. Nature Protocols, 2(11), 2896-2902.
7.	
Madin, K.; Sawasaki, T.; Ogasawara, et al. 2000. A highly efficient and robustcell-free protein synthesis system prepared from wheat embryos: plants apparently
contain a suicide system directed at ribosomes. Proc Natl Acad Sci U S A, 97(2),559-564.
8.	
Shimizu, Y.; Inouse, A.; Tomari, Y.; et al. 2001. Cell-free translation reconstituted
with purified components. Nat Biotechnol, 19(8), 751-755.

| Stable Isotopes for Biomolecular NMR

isotope.com

Cell-Free Protein Expression
CIL offers a wide variety of products for cell-free protein expression.
Cell-free protein expression methods offer several advantages
over expression using E. coli or other in vivo expression systems.
These advantages include increased speed, ability to express toxic
proteins, ease of amino acid type selective labeling, and an open
system that allows cofactors, chaperones, redox molecules, anddetergents to be easily be added to the expression system. Cell-freemethods also allow co-expression of multiple proteins and are
amenable to automation.
CIL is pleased to distribute a wide range of products from CellFree
Sciences. CIL also offers algal-derived amino acid mixes andconveniently packaged sizes of individual crystalline amino acids.
Profiles for
16 Amino Acid Mixture(16 AA)

Profiles for
20 Amino Acid Mixture(20 AA)

Approximate percentages, subject to lot-to-lot variability.
L-Alanine7%

L-Alanine6%

L-Arginine
L-Aspartic acid
L-Glutamic acid
Glycine
L-Histidine
L-Isoleucine
L-Leucine
L-Lysine
L-Methionine
L-Phenylalanine
L-Proline
L-Serine
L-Threonine
L-Tyrosine
L-Valine

7%
10%
10%
6%
2%
4%
10%
14%
1%
4%
7%
4%
5%
4%
5%

L-Arginine
L-Asparagine
L-Aspartic acid
L-Cysteine
L-Glutamic acid
L-Glutamine
Glycine
L-Histidine
L-Isoleucine
L-Leucine
L-Lysine
L-Methionine
L-Phenylalanine
L-Proline
L-Serine
L-Threonine
L-Tryptophan
L-Tyrosine
L-Valine

6%
5%
8%
3%
9%
5%
5%
1%
3%
9%
12%
1%
4%
5%
4%
4%
3%
3%
4%

Amino Acid Mixes for Cell-Free Protein Expression
Catalog No.
CLM-1548
DLM-2082
NLM-2161
CNLM-452

Description
Algal amino acid mixture (16AA) (13C, 97-99%)
Algal amino acid mixture (16AA) (D, 98%)
Algal amino acid mixture (16AA) (15N, 98%)
Algal amino acid mixture (16AA)(13C, 97-99%; 15N, 97-99%)
DNLM-819
Algal amino acid mixture (16AA) (D, 98%; 15N, 98%)
CDNLM-2496 Algal amino acid mixture (16AA)(13C, 97-99%; D, 97-99%; 15N, 97-99%)
ULM-2314
Algal amino acid mixture (16AA) (unlabeled)
DLM-6819“Cell free” amino acid mix (20AA) (D, 98%)
NLM-6695“Cell free” amino acid mix (20AA) (15N, 96-98%)
CNLM-6696“Cell free” amino acid mix (20AA)(13C, 97-99%; 15N, 97-99%)
DNLM-6818“Cell free” amino acid mix (20AA)(D, 98%; 15N, 98%)
CDNLM-6784 “Cell free” amino acid mix (20AA)(13C, 97-99%; 15N, 97-99%; D, 97-99%)
ULM-7891“Cell free” amino acid mix (20AA) (unlabeled)➤ 25 mg and 50 mg packaged sizes are available for all CIL
amino acids. Experience the convenience and flexibility ofusing individually packaged crystalline amino acids withyour cell-free protein expression system.* See page 28
for a complete listing of CIL’s free amino acids.
*CIL does not provide protocols in formulation of amino acid mixtures,as the formulations may vary depending on application and reaction
scale. The pH should be checked prior to use for first-time amino acidformulations.

❛❛We have been a loyal customer of CIL for stable isotopelabeled products for our research in the study of proteins.
CIL has provided reliable, in-stock, high-quality products for
our lab. We have enjoyed a nice working relationship with
CIL over the years. ❜❜Ravi Pratap Barnwal, PhD
UGC Assistant Professor
Department of Biophysics
Panjab University

cilsales@isotope.com | intlsales@isotope.com | isotope.com 21

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Cell-Free Protein Expression
CellFree Sciences (CFS) Products and Kits for Cell-Free Protein Expression
Producing proteins at will, often a bottleneck in postgenome
studies, has become a reality with the advent of the robust wheat
germ cell-free protein expression system. CellFree Sciences’
ENDEXT® wheat germ cell-free system permits both highthroughput protein screening and microgram- to milligram-scaleprotein production overnight. Protein synthesis protocols for the
ENDEXT system have been optimized for a wide range of proteins.
They have also been incorporated into robotic protein synthesizers,
versatile Protemist® DT II and mass-producing Protemist XE. Being
eukaryotic and free from physiological constraints that hamper invivo systems, the wheat germ cell-free system allows synthesis, withor without additives, of a broad spectrum of protein and protein
complexes ranging from 10 kDa to 360 kDa in well-folded andsoluble forms. Most importantly, labeled protein samples canbe easily produced for NMR studies when labeled amino acids
are used.

Catalog No.

Description

Product Name

Amount

CIL is pleased to offer a range of CFS products and kits. Pleasecontact CFS directly (tech-sales@cfsciences.com) if you would
like to use CFS’ lab services to prepare a pEU plasmid with yourtarget gene sequence, characterize the yield and solubility ofyour expressed protein, or produce a prescribed amount ofprotein using the wheat germ cell-free system.
ENDEXT® is a registered trademark of CellFree Sciences.
Protemist® is a registered trademark of Emerald BioSystems.
CIL is a distributor of the above-referenced CFS products in the US and Europe.

Expression Vectors for Use in Wheat Germ Cell-Free Protein Expression System
CFS-PEU-v1.0

pEU Vector Set

19 vectors

Vector set for expression of proteins in the wheat germ system: 19 different
vectors to work with His-, GST-, and FLAG-tag, untagged proteins.
Set includes positive controls and vector for FLEXIQuant method.

Coupled Cell-Free in Vitro Transcription and Translation Reactions for Protein Research Applications
CFS-EDX-ONE

Premium ONE Expression Kit

24 reactions

Coupled in vitro transcription and translation reaction format for rapid
protein expression experiments enabling protein analysis by different
detection methods such as SDS-PAGE, Western Blot Analysis, or working
with labeled proteins. Kit uses bilayer reaction on 55 µL scale.

Linked Cell-Free Protein Expression Reactions for Protein Research Applications (Bilayer Format)
Core kits with individual reagents for linked cell-free transcription and translation reactions on different reaction scales
CFS-C7WEPRO7240 Core Kit
1 mL extract
SMALL reaction scale of 227 µL (up to 100 reactions), a MEDIUM reaction
scale of 1.2 mL (up to 20 reactions), and a LARGE reaction scale of 6 mL
(up to 4 reactions).
CFS-C7GWEPRO7240G Core Kit
1 mL extract
SMALL reaction scale of 227 µL (up to 100 reactions), a MEDIUM reaction
scale of 1.2 mL (up to 20 reactions), and a LARGE reaction scale of 6 mL
(up to 4 reactions). Kit uses precleared extract intended for preparing
GST-fusion proteins.
CFS-C7HWEPRO7240H Core Kit
1 mL extract
SMALL reaction scale of 227 µL (up to 100 reactions), a MEDIUM reaction
scale of 1.2 mL (up to 20 reactions), and a LARGE reaction scale of 6 mL
(up to 4 reactions). Kit uses precleared extract intended for preparing
His-tagged fusion proteins.
CFS-C8WEPRO8240 Core Kit
1 mL extract
WEPRO8240 is WEPRO7240 without amino acids to enable protein labelingreactions. SMALL reaction scale of 240 μL (up to 100 reactions), a MEDIUM
reaction scale of 1.2 mL (up to 20 reactions), and a LARGE reaction scaleof 6 mL (up to 4 reactions).
CFS-C8GWEPRO8240G Core Kit
1 mL extract
WEPRO8240G is WEPRO7240G without amino acids to enable proteinlabeling reactions. SMALL reaction scale of 240 μL (up to 100 reactions),a MEDIUM reaction scale of 1.2 mL (up to 20 reactions), and a LARGE
reaction scale of 6 mL (up to 4 reactions). Kit uses precleared extract
intended for preparing GST-fusion proteins.
CFS-C8HWEPRO8240H Core Kit
1 mL extract
WEPRO8240H is WEPRO7240H without amino acids to enable proteinlabeling reactions. SMALL reaction scale of 240 μL (up to 100 reactions),a MEDIUM reaction scale of 1.2 mL (up to 20 reactions), and a LARGE
reaction scale of 6 mL (up to 4 reactions). Kit uses precleared extract
intended for preparing His-tagged fusion proteins.

22

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| Stable Isotopes for Biomolecular NMR

isotope.com

Cell-Free Protein Expression
Catalog No.

Product Name

AmountDescription

Linked Cell-Free Protein Expression Reactions for Protein Research Applications (Bilayer Format) continued
Premixed reagents for linked cell-free transcription and translation reaction on 227 µL scale
CFS-EDX-PLUS
Premium PLUS Expression Kit
8 reactions
Transcription and translation reagents are premixed for easy handling.
Kit uses bilayer reaction on 227 µL scale. This kit contain an expression
vector, a positive control, and a PCR primer set.
CFS-PRK-S16
Protein Research Kit S16
16 reactions
Transcription and translation reagents are premixed for easy handling.
Kit uses bilayer reaction on 227 µL scale.
CFS-PRK-G16
Protein Research Kit G16
16 reactions
Premixed transcription and translation reagents for GST-fusion protein
expression. Kit uses bilayer reaction on 227 µL scale.
CFS-PRK-H16
Protein Research Kit H16
16 reactions
Premixed transcription and translation reagents for His-fusion protein
expression. Kit uses bilayer reaction on 227 µL scale.
Linked Cell-Free Protein Expression Reactions for Large Protein Needs in Research/Product Development/Production (Dialysis Format)
CFS-D7WEPRO7240 Dialysis Kit

1 mL extract

CFS-D7GWEPRO7240G Dialysis Kit

1 mL extract

CFS-D7HWEPRO7240H Dialysis Kit

1 mL extract

Dialysis kit provides reagents to perform dialysis-driven expression reactions
on a 50 μL small-scale (up to 100 reactions) or preparative 3 mL scale(up to 2 reactions).
Dialysis kit provides reagents to perform dialysis-driven expression reactions
on a 50 μL small-scale (up to 100 reactions) or preparative 3 mL scale(up to 2 reactions). Kit uses precleared extract intended for preparing
GST-fusion proteins.
Dialysis kit provides reagents to perform dialysis-driven expression reactions
on a 50 μL small-scale (up to 100 reactions) or preparative 3 mL scale(up to 2 reactions). Kit uses precleared extract intended for preparing
His-tagged fusion proteins.

Special Applications
Protein labeling for protein mass spectrometry
CFS-FLEX-MS
FLEXIQuant PLUS Expression Kit

8 reactions

CFS-FLEX-PEP

FLEX-Tag Peptide

8 vials

CFS-PRK-MS

Protein Research Kit for MS

16 reactions

CFS-C8MSWEPRO8240 MS Core Kit

1 mL extract

CFS-C8GMSWEPRO8240G MS Core Kit

1 mL extract

CFS-C8HMS

WEPRO8240H MS Core Kit

1 mL extract

Protein synthesis kit with 13C/ 15N labeled lysine and arginine for MRM/SRM
mass spectrometry. Kit uses bilayer reaction on 227 μl scale. This kit provides
expression vector and peptide to conduct FLEXIQuant experiments for using
the FLEX-tag for quantifying the reference standard.
Lyophilized, synthetic FLEX-tag peptide (TENLYFQGDISR); >95% peptide
purity, 1 nmol dried peptide per vial.
Protein synthesis kit with 13C/ 15N labeled lysine and arginine for MRM/SRM
mass spectrometry. Kit uses bilayer reaction on 227 µL scale.
Protein synthesis kit with 13C/ 15N labeled lysine and arginine for MRM/SRM
mass spectrometry. SMALL reaction scale of 240 μL (up to 100 reactions),a MEDIUM reaction scale of 1.2 mL (up to 20 reactions), and a LARGE
reaction scale of 6 mL (up to 4 reactions).
Protein synthesis kit with 13C/ 15N labeled lysine and arginine for MRM/SRM
mass spectrometry. SMALL reaction scale of 240 μL (up to 100 reactions),a MEDIUM reaction scale of 1.2 mL (up to 20 reactions), and a LARGE
reaction scale of 6 mL (up to 4 reactions). Kit uses precleared extract
intended for preparing GST-fusion proteins.
Protein synthesis kit with 13C/ 15N labeled lysine and arginine for MRM/SRM
mass spectrometry. SMALL reaction scale of 240 μL (up to 100 reactions),a MEDIUM reaction scale of 1.2 mL (up to 20 reactions), and a LARGE
reaction scale of 6 mL (up to 4 reactions). Kit uses precleared extract
intended for preparing His-tagged fusion proteins.

Continued ➤cilsales@isotope.com | intlsales@isotope.com | isotope.com 23

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Cell-Free Protein Expression
Catalog No.

Product Name

AmountDescription

Protein labeling for NMR
CFS-C8N
WEPRO8240 (15N) Core Kit

1 mL extract

CFS-C8GNWEPRO8240G (15N) Core Kit

1 mL extract

CFS-C8HNWEPRO8240H (15N) Core Kit

1 mL extract

CFS-C8CNWEPRO8240 (13C/ 15N) Core Kit

1 mL extract

CFS-C8GCNWEPRO8240G (13C/ 15N) Core Kit

1 mL extract

CFS-C8HCNWEPRO8240H (13C/ 15N) Core Kit

1 mL extract

SMALL reaction scale of 240 μL (up to 100 reactions), a MEDIUM reaction
scale of 1.2 mL (up to 20 reactions), and a LARGE reaction scale of 6 mL
(up to 4 reactions). The translation buffer contains stable isotope 15N-labeledamino acids.
SMALL reaction scale of 240 μL (up to 100 reactions), a MEDIUM reaction
scale of 1.2 mL (up to 20 reactions), and a LARGE reaction scale of 6 mL
(up to 4 reactions). Kit uses precleared extract intended for preparing
GST-fusion proteins. The translation buffer contains stable isotope
15
N-labeled amino acids.
SMALL reaction scale of 240 μL (up to 100 reactions), a MEDIUM reaction
scale of 1.2 mL (up to 20 reactions), and a LARGE reaction scale of 6 mL
(up to 4 reactions). Kit uses precleared extract intended for preparing
His-tagged fusion proteins. The translation buffer contains stable isotope
15
N-labeled amino acids.
SMALL reaction scale of 240 μL (up to 100 reactions), a MEDIUM reaction
scale of 1.2 mL (up to 20 reactions), and a LARGE reaction scale of 6 mL
(up to 4 reactions). The translation buffer contains stable isotope 13C- and15
N-labeled amino acids.
SMALL reaction scale of 240 μL (up to 100 reactions), a MEDIUM reaction
scale of 1.2 mL (up to 20 reactions), and a LARGE reaction scale of 6 mL
(up to 4 reactions). Kit uses precleared extract intended for preparing
GST-fusion proteins. The translation buffer contains stable isotope
13
C- and 15N-labeled amino acids.
SMALL reaction scale of 240 μL (up to 100 reactions), a MEDIUM reaction
scale of 1.2 mL (up to 20 reactions), and a LARGE reaction scale of 6 mL
(up to 4 reactions). Kit uses precleared extract intended for preparing
His-tagged fusion proteins. The translation buffer contains stable isotope
13
C- and 15N-labeled amino acids.

Special Applications – continued

Expression of membrane proteins in proteoliposomes
CFS-EDX-PLUS-PLE
Proteoliposome Plus
Expression Kit

8 reactions

CFS-CPLE-BDL

6 reactions

Proteoliposome BD
Expression Kit-L

CFS-ASL

Asolectin Liposomes(lyophilized)
CFS-ASL-BT
Biotinylated Liposomes(lyophilized)
Expression of proteins with disulfide bonds
CFS-C7-DTT
WEPRO7240-DTT Core Kit

1 set
1 set

1 mL extract

The reagents in this kit are DTT free. This allows users to reduce DTT
concentrations for improved stability of S-S bonds. SMALL reaction
scale of 240 μL (up to 100 reactions), a MEDIUM reaction scale of 1.2 mL(up to 20 reactions), and a LARGE reaction scale of 6 mL (up to 4 reactions).
Note: reducing the DTT concentration will commonly reduce protein yields
in the translation reactions.
Introduction into protein expression doing an in vitro transcription andtranslation experiment. For one course with 20 students including plastic
consumables for use at schools.
Introduction into protein expression doing an in vitro transcription andtranslation experiment. For one course with 20 students. This version
does not provide plastic consumables.
Tube rack, floater, orange filter, blue LED light, UV – LED light.

Teaching materials
CFS-EDU-P

Protein Expression Reagent Kit

10 experiments

CFS-EDU-N

Protein Expression Reagent Kit

10 experiments

CFS-EDU-E

Laboratory Instrument Set

For one course,
reusable

24

Starter kit for easy expression of membrane proteins on 240 μL bilayer
reaction scale. Premixed reagents already contain liposomes for easy
preparation of proteoliposome complexes.
For high-yield expression of membrane proteins on 2.5 mL BD reaction scalefor direct formation of proteoliposomes. Additional reagents provided fortesting expression vector in absence of liposomes.
Set with 6 vials each holding 10 mg of lyophilized liposomes prepared fromasolectin (mixture of phospholipids from soybean).
1 vial holding 10 mg of lyophilized liposomes prepared from asolectin(mixture of phospholipids from soybean) and added biotin.

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| Stable Isotopes for Biomolecular NMR

isotope.com

Cell-Free Protein Expression
Catalog No.

Product Name

AmountDescription

80,000 units
1 mL
2,400 units
30 μL
1,200 units
15 μL
1 mL
1 mL
1 set

Enzyme for mRNA synthesis and RNase inhibitor

1 mL
1 mL

High-performance wheat germ extract
Precleared high-performance wheat germ extract for working with
GST-tagged proteins
Precleared high-performance wheat germ extract for working with
His-tagged proteins
High-performance wheat germ extract for labeling, containing only tracesof amino acids; WEPRO7240 without amino acids
High-performance wheat germ extract for labeling, containing only tracesof amino acids, preprocessed for GST tag affinity purification; WEPRO7240G
without amino acids
High-performance wheat germ extract for labeling, containing only tracesof amino acids, preprocessed for His tag affinity purification; WEPRO7240H
without amino acids
Translation buffer; to be used with WEPRO7240 series
Translation buffer; to be used with WEPRO7240 series
Translation buffer for labeling; SUB-AMIX SGC without amino acids;
to be used with WEPRO8240 series
Translation buffer; to be used with WEPRO7240 series on Protemist XE
Translation buffer for labeling; SUB-AMIX SG without amino acids;
to be used with WEPRO8240 series on Protemist XE

Individual Reagents
Transcription reagents
CFS-TSC-ENZ
SP6 RNA Polymerase plus
RNase Inhibitor
CFS-TSC-ENZ-S
SP6 RNA Polymerase plus
RNase Inhibitor
CFS-TSC-ENZ-S/2
SP6 RNA polymerase plus
RNase Inhibitor
CFS-TSC-NTP
NTP Mix
CFS-TSC-5TB-LM
5x Transcripton Buffer LM
CFS-TB
Transcription Set(additional set)
Translation reagents
CFS-WGE-7240
WEPRO7240
CFS-WGE-7240G
WEPRO7240G

Enzyme for mRNA synthesis and RNase inhibitor
Enzyme for mRNA synthesis and RNase inhibitor
25 mM mixture of ATP, UTP, GTP, and CTP
Transcription buffer for WEPRO7240/8240 series
Set contains SP6 RNA polymerase (1,200 units/15 μL), RNase inhibitor(15 μL), 25 mM NTPs (120 μL), and 5x transcription buffer LM (240 μL)

CFS-WGE-7240H

WEPRO7240H

1 mL

CFS-WGE-8240

WEPRO8240

1 mL

CFS-WGE-8240G

WEPRO8240G

1 mL

CFS-WGE-8240H

WEPRO8240H

1 mL

CFS-SUB-SGC
CFS-SUB
CFS-SUB-SGC-NA

SUB-AMIX SGC
SUB-AMIX SGC SUB Set
SUB-AMIX SGC NA

12.5 mL × 4
7 mL × 4
12.5 mL × 4

CFS-SUB-SG
CFS-SUB-SG-NAA

SUB-AMIX SG
SUB-AMIX SG NA

12.5 mL × 4
12.5 mL × 4

Reaction Cups
Reaction Cups
Reaction Cups
Robotic Tips (bulk)
Robotic Tips (racked)

1 × 6 cups
10 × 6 cups
50 × 6 cups
2 × 500 tips
24 × 96 tips

6 × disposable reaction cups
60 × disposable reaction cups
300 × disposable reaction cups
1000 × disposable pipetting tips
24 × ready-to-use disposable pipetting tips in 96-well racks

Tubes

2 × 500 tubes

1000 × 2 mL tubes

Preassembled Kit
Preassembled Kit
Connectors

1 set
1 set
1 set

10 × 10 mL reactions
10 × 20, 30, 40 mL reactions
24 × Vivaflow connector (needed for 30 and 40 mL reactions)

Consumables
Protemist® DT II
CFS-DT2-P01-1
CFS-DT2-P01-10
CFS-DT2-P01-50
CFS-ASP-DIP1000V
CFS-ASPDIP1000V-PT24
CFS-DT2-P02-2
Protemist XE
CFS-XE-P10
CFS-XE-P20
CFS-XE-VFC

cilsales@isotope.com | intlsales@isotope.com | isotope.com 25

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Selective Amino Acid-Type Labeling
Selective amino acid-type labeling is used to simplify crowded
spectra in order to aid in spectral interpretation or to provide
specific probes for dynamic and structural studies. This techniquerequires the addition of labeled amino acids (~50-250 mg/L)
to either minimal or rich growth media prior to protein induction.

The cells will generally utilize the supplemented amino acids forprotein synthesis prior to undergoing the de novo synthesis of thetarget amino acids. Please see page 28 for a complete listing ofamino acids that may be used with either in vivo growth systems
or cell-free protein expression methods.

Selective Isotope-Labeling Methods for Protein Structural Studies

Investigator
Spotlight

Hanudatta S. Atreya, PhD
NMR Research Centre, Indian Institute of Science, Bangalore, India
One of the major contributing factors to the rapid advance of
biomolecular NMR spectroscopy is the emergence of different
isotope-labeling methods. Recent developments in biotechnology
have made it easier and economical to introduce 13C,15N and 2H
into proteins and nucleic acids. At the same time, there has been
an explosion in the number of NMR experiments that utilize suchisotope-labeled samples. Thus, a combination of isotopic labelingand multidimensional, multinuclear experiments has significantly
expanded the range of problems in structural biology amenable
to NMR.
Isotope labeling in proteins can be broadly classified into four
categories: uniform, amino acid-type selective, site-specific,
and random/fractional labeling. The beginning of systematizedisotope labeling in proteins can be traced back to late 60s in thegroup of Jardetsky and Katz and coworkers.1,2 Theirs was also oneof the first amino acid-type selective-labeling methods involving
incorporation of specific protonated amino acids against a
deuterated background. In the 80s uniform (13C/ 15N) and selective
incorporation of 15N-labeled amino acids against an unlabeled(12C / 14N) background was developed.3 Subsequently, a varietyof labeling methods have emerged (reviewed in [4] and [5]and illustrated in Figure 1).

Uniform labeling(13C/ 15N/ 2H)
Amino acidselective unlabeling/
protonation

Amino acidselective labeling

Segmental
labeling

Isotopelabeling

Methyl-specificprotonation

Perdeuteration/
random fractional
deuteration

Site-specificlabeling
Stereo-arrayedisotope labeling

Figure 1. Different isotope-labeling methods.

26

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In addition to uniform (13C/ 15N/ 2H) labeling, amino acid-type
or site-selective labeling is often pursued as it helps in spectral
simplification and provides specific probes for structural anddynamic studies. Selective amino acid-type labeling also aids insequence-specific resonance assignments by helping to identify
resonances which are otherwise buried in the crowded regionsof 2D and 3D NMR spectra. However, a disadvantage of thismethod is the possible mis-incorporation of 15N label in undesired
amino acids (also called as “isotope scrambling”).3 This happens
due to metabolic conversion of one amino acid to another in thebiosynthetic pathway of the cell. The problem becomes more
severe for amino acids or intermediates higher up in the metabolic
pathway such as Asp, Glu and Gln (See Figure 2 showing thebiosynthetic pathway in E. coli ). For those which are end-productsin the production pipeline (Ala, Arg, Asn, Cys, His, Ile, Lys, Met,
Pro, and Trp), isotope scrambling is minimal, and the remaining(Gly, Phe, Leu, Ser, Thr, Tyr, and Val) have medium to weakinterconversion. Isotope scrambling in E. coli can be minimized by
reducing the activity of the enzyme(s) catalyzing the interconversion
or amino acid transfer using either specific (auxotrophic) strains3
or using enzyme inhibitors.6 Another alternative is to use cell-freeor in vitro expression systems which lack these enzymes.4

Selective amino acid-type labeling also aids insequence-specific resonance assignments by helping
to identify resonances which are otherwise buried in
the crowded regions of 2D and 3D NMR spectra.
One drawback of amino acid selective labeling is the expense
associated with the use of 13C/ 15N labeled amino acids. A relatively
inexpensive method is that of amino acid selective “unlabeling” orreverse labeling. In this method, the host organism is grown on a
medium containing the desired unlabeled (i.e., 1H/ 12C/ 14N) aminoacid against a labeled (13C/ 15N) background. This is somewhat akinto the selective protonation experiment by Jardetsky1 and Katz.2
Reverse labeling was first used by Bax and coworkers7 anddeveloped further by other groups for different applications.8,9,10
The problem of isotope scrambling (in this case being the misincorporation of 14N) remains largely the same as in the selectivelabeling approach mentioned above (for a detailed table of possible
scrambling of 14N see reference 10).

| Stable Isotopes for Biomolecular NMR

isotope.com

Selective Isotope-Labeling Methods for Protein Structural Studies (continued)

GlycerolGlucoseGlucose-6-phosphate

Phe
Tyr
Trp

Chorismate

Shikimate

Asn

Phosphenol-pyruvate

Cys

Gly

Pyruvate

α-Ketoisovalerate

AcetateAcetyl CoA

Val Leu

Asp

Homoserine

α-Ketobutyrate

Ser

Ala

Oxaloacetate

Isocitrate

Malateα-Ketoglutorate

Aspartate Semi-aldehyde
Lys

Glucose-3-phosphate

Met

Succinate

Glu

Gln Pro Arg

Thr

Ile

Figure 2. Amino acid biosynthesis in E. coli.

In addition to the above, new isotope-labeling methods continue
to be developed. Additional methods such as segmental labeling11
and stereo-arrayed isotope labeling (SAIL)12 have opened up new

avenues in protein structural studies. The future points toward
a combination of different isotope-labeling methods to address
challenging and complex problems in structural biology.

References
1.	Markley, J.K.; Putter, I.; Jardetsky, O. 1968. High-resolution nuclear magnetic
resonance spectra of selectively deuterated staphylococcal nuclease. Science,
161(3847),1249-1251.
2.	Crespi, H.L.; Rosenberg, R.M.; Katz, J.L. 1968. Proton magnetic resonance ofproteins fully deuterated except for H-leucine side chains. Science, 161(3843),
795-796.
3.	Muchmore D.D.; McIntosh, L.P.; Russell, C.B.; et al. 1989. Expression andnitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic
resonance. Methods Enzymol, 177, 44-73.
4.	Ohki, S.; Kainosho, M. 2008. Stable isotope labeling for protein NMR.
Prog NMR Spectrosc, 53, 208-226.
5.	Lu, K.; Miyazaki, Y.; Summers, M.F. 2010. Isotope labeling strategies for NMR
studies of RNA. J Biomol NMR, 46(1), 113-125.

8.	Shortle, D. 1995. Assignment of amino acid type in 1H-15N correlation spectraby labeling with 14N amino acids. J Magn Reson B, 105(1), 88-90.
9.	Atreya, H.S.; Chary, K.V.R. 2001. Selective “unlabeling” of amino acids infractionally 13C-labeled proteins: An approach for stereospecific NMR assignmentsof CH3 groups in Val and Leu residues. J Biomol NMR, 19(3), 267-272.
10.	Krishnarjuna, B.; Jaiupuria, G.; Thakur, A.D.; et al. 2010. Amino acid selective
unlabeling for sequence specific resonance assignments in proteins. J Biomol NMR,
49(1), 39-51.
11.	Cowburn, D. and Muir, T.W. 2001. Segmental isotopic labeling for structural
biological applications of NMR. Methods Enzymol, 339, 41–54.
12.	Takeda, M.; Sugimori, N.; Torizawa, T.; et al. 2008. Structure of the putative
32 kDa myrosinase binding protein from arabidopsis (At3g16450.1) determinedby SAIL-NMR. FEBS J, 275(23), 5873-5884.

6.	Tong, K.I.; Yamamoto, M.; Tanaka, T. 2008. A simple method for amino acidselective isotope labeling of recombinant proteins in E. coli. J Biomol NMR, 42(1),59-67.
7.	Vuister, G.W.; Kim, S.J.; Wu, C.; et al. 1994. 2D and 3D NMR study ofphenylalanine residues in proteins by reverse isotopic labeling. J Am Chem Soc,
116(20), 9206-9210.

cilsales@isotope.com | intlsales@isotope.com | isotope.com 27

Cambridge Isotope Laboratories, Inc.

| Stable Isotopes for Biomolecular NMR

Amino Acids
CIL offers a comprehensive list of labeled amino acids for use in a variety of NMR investigations. The large number of available labelingschemes maximizes flexibility involved in designing experiments to probe structure, dynamics and binding of biological macromolecules.
A number of package sizes are available, including 25 mg and 50 mg, which are convenient for use in cell-free protein expression.
Pricing and availability can be found on isotope.com. If a specific size is not listed, custom packaging may be available; please inquire.
Catalog No.
CLM-8755
CLM-8756
NLM-1656
CNLM-3440
CNLM-8457
CNLM-3946
CLM-116
CLM-2016
CLM-117
CLM-2734
CLM-2184-H
DLM-3101
DLM-248
DLM-250
DLM-251
NLM-454
CDLM-8649
CDLM-3439
CNLM-6993
CNLM-3594
CNLM-534-H
DNLM-7178
CDNLM-6800
CLM-2070
CLM-1268
CLM-2051
CLM-2265-H

Descriptionb-Alanine (3-13C, 99%)b-Alanine (13C3, 99%)b-Alanine (15N, 98%)b-Alanine (3-13C, 99%; 15N, 98%)b-Alanine (1,2-13C2, 99%; 15N, 98%)b-Alanine (13C3, 98%; 15N, 96-99%)
L-Alanine (1-13C, 99%)
L-Alanine (2-13C, 99%)
L-Alanine (3-13C, 99%)
L-Alanine (2,3-13C2, 99%)
L-Alanine (13C3, 99%)
L-Alanine (2-D, 96-98%)
L-Alanine (3,3,3-D3, 99%)
L-Alanine (2,3,3,3-D4, 98%)
L-Alanine (D7, 98%)
L-Alanine (15N, 98%)
L-Alanine (3-13C, 99%; 2-D, 96%)
L-Alanine (3-13C, 99%; 3,3,3-D3, 98%)
L-Alanine (1-13C, 99%; 15N, 98%)
L-Alanine (2-13C, 99%; 15N, 98%)
L-Alanine (13C3, 99%; 15N, 99%)
L-Alanine (2,3,3,3-D4, 98%; 15N, 98%)
L-Alanine (13C3, 97-99%; D4, 97-99%; 15N, 97-99%)
L-Arginine·HCl (guanido-13C, 99%)
L-Arginine·HCl (1-13C, 99%)
L-Arginine·HCl (1,2-13C2, 99%)
L-Arginine·HCl (13C6, 99%)

DLM-6038
DLM-541
NLM-1267
NLM-395
NLM-396
CDLM-3789
CNLM-7819
CNLM-539-H

L-Arginine·HCl (4,4,5,5-D4, 94%)
L-Arginine·HCl (D7, 98%)
L-Arginine·HCl (a-15N, 98%)
L-Arginine·HCl (guanido-15N2, 98%)
L-Arginine·HCl (15N4, 98%)
L-Arginine·HCl (5-13C, 99%; 4,4,5,5-D4, 95%)
L-Arginine·HCl (1-13C, 99%; a-15N, 98%)
L-Arginine·HCl (13C6, 99%; 15N4, 99%)

DNLM-7543
CDNLM-6801
ULM-8347
CLM-8699-H
DLM-6844
NLM-120
NLM-3286
CNLM-7818
CNLM-3819-H
DNLM-6932
CDNLM-6802

L-Arginine·HCl (D7, 98%; 15N4, 98%)
L-Arginine·HCl (13C6, 97-99%; D7, 97-99%; 15N4, 97-99%)
L-Arginine·HCl (unlabeled)
L-Asparagine·H2O (13C4, 99%)
L-Asparagine·H2O (2,3,3-D3, 94%)
L-Asparagine·H2O (amide-15N, 98%)
L-Asparagine·H2O (15N2, 98%)
L-Asparagine·H2O (1,4-13C2, 99%; a-15N, 98%)
L-Asparagine·H2O (13C4, 99%; 15N2, 99%)
L-Asparagine·H2O (2,3,3-D3, 98%; 15N2, 98%)
L-Asparagine·H2O (13C4, 97-99%; D3, 97-99%; 15N2, 97-99%)

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

28

tel: +1.978.749.8000 | 1.800.322.1174 (USA) | fax: +1.978.749.2768

Unit Size
Please inquire
Please inquire
0.25 gPlease inquire
Please inquire
0.25 g
0.5 g, 1 g
0.1 g, 0.25 g, 0.5 g
0.5 g, 1 g
0.25 g, 0.5 g
0.1 g, 0.25 g, 0.5 g
Please inquire1g
0.1 g, 1 g
1g
0.5 g, 1 g
1g
Please inquire
0.25 g0.25 g
0.1 g, 0.25 g, 0.5 g,
0.25 g, 0.5 g
0.25 g
0.5 g
0.1 g
0.1 g
0.05 g, 0.1 g, 0.25 g,
0.5 g, 1 g
Please inquire
0.1 g
Please inquire
0.5 g, 1 g
0.1 g
Please inquire
Please inquire
0.05 g, 0.1 g, 0.25 g,
0.5 g, 1 g
0.25 g0.25 g0.05 g, 0.1 g
0.05 g0.1 g0.25 g, 0.5 g
0.25 g, 0.5 g
0.25 g
0.1 g, 0.25 g, 0.5 g
0.25 g0.25 g| Stable Isotopes for Biomolecular NMR

isotope.com

Catalog No.

Description

CLM-3616
CLM-3617
CLM-627
CLM-519
CLM-4455
CLM-1801-H
DLM-546
NLM-718
CNLM-7817
CNLM-544-H
DNLM-6931
CDNLM-6803
CLM-3852
CLM-1868
CLM-4320-H
DLM-769
DLM-6901
NLM-2295
CNLM-7815
CNLM-3871-H
DNLM-6902
CDNLM-6809
CLM-520
DLM-9812
NLM-3818
CNLM-4244-H
CDNLM-8659
CLM-674
CLM-2474
CLM-4742
CLM-2431
CLM-613
CLM-2024
CLM-3646
CLM-1800-H
DLM-3725
DLM-556
NLM-135
CNLM-7812
CNLM-554-H
DNLM-6996
CDNLM-6804
OLM-8028
CLM-3612
CLM-3613
CLM-770
CLM-1166
CLM-2001
CLM-3641
CLM-1822-H
DLM-1826
NLM-1016

L-Aspartic acid (1-13C, 99%)
L-Aspartic acid (2-13C, 99%)
L-Aspartic acid (3-13C, 98-99%)
L-Aspartic acid (4-13C, 99%) CP 96%
L-Aspartic acid (1,4-13C2, 99%)
L-Aspartic acid (13C4, 99%)
L-Aspartic acid (2,3,3-D3, 98%)
L-Aspartic acid (15N, 98%)
L-Aspartic acid (1,4-13C2, 99%; 15N, 98%)
L-Aspartic acid (13C4, 99%; 15N, 99%)
L-Aspartic acid (2,3,3-D3, 98%; 15N, 98%)
L-Aspartic acid (13C4, 97-99%; D3, 97-99%; 15N, 97-99%)
L-Cysteine (1-13C, 99%)
L-Cysteine (3-13C, 99%)
L-Cysteine (13C3, 99%)
L-Cysteine (3,3-D2, 98%)
L-Cysteine (2,3,3-D3, 98%)
L-Cysteine (15N, 98%)
L-Cysteine (1-13C, 99%; 15N, 98%)
L-Cysteine (13C3, 99%; 15N, 99%)
L-Cysteine (2,3,3-D3, 98%; 15N, 98%)
L-Cysteine (13C3, 97-99%; D3, 97-99%; 15N, 97-99%)
L-Cystine (3,3’-13C2, 99%)
L-Cystine (3,3,3’,3’-D4, 98%)
L-Cystine (15N2, 98%)
L-Cystine (13C6, 99%; 15N2, 99%)
L-Cystine (13C6, 98%; D6, 98%; 15N2, 98%) CP 95%
L-Glutamic acid (1-13C, 99%)
L-Glutamic acid (2-13C, 99%)
L-Glutamic acid (3-13C, 99%)
L-Glutamic acid (4-13C, 98-99%)
L-Glutamic acid (5-13C, 99%)
L-Glutamic acid (1,2-13C2, 99%)
L-Glutamic acid (3,4-13C2, 99%)
L-Glutamic acid (13C5, 99%)
L-Glutamic acid (2,4,4-D3, 97-98%)
L-Glutamic acid (2,3,3,4,4-D5, 98%)
L-Glutamic acid (15N, 98%)
L-Glutamic acid (1-13C, 99%; 15N, 98%)
L-Glutamic acid (13C5, 99%; 15N, 99%)
L-Glutamic acid (2,3,3,4,4-D5, 98%; 15N, 98%)
L-Glutamic acid (13C5, 97-99%; D5, 97-99%; 15N, 97-99%)
L-Glutamic acid·HCl (17O4, ~30%)
L-Glutamine (1-13C, 99%)
L-Glutamine (2-13C, 99%)
L-Glutamine (4-13C, 99%)
L-Glutamine (5-13C, 99%)
L-Glutamine (1,2-13C2, 99%)
L-Glutamine (3,4-13C2, 99%)
L-Glutamine (13C5, 99%)
L-Glutamine (2,3,3,4,4-D5, 97%)
L-Glutamine (a-15N, 98%)

Unit Size
Please inquire
Please inquire
0.05 g, 0.1 g, 0.25 g
Please inquire
0.5 g
0.25 g, 0.5 g, 1 g
0.1 g, 0.25 g
0.5 g, 1 g
0.25 g0.25 g, 0.5 g, 1 g
0.25 g, 0.5 g
0.25 g0.5 g0.25 g
0.1 g
0.1 g
0.1 g
0.25 gPlease inquire
0.1 g, 0.25 g
0.25 g0.25 g0.25 g0.5 g0.25 gPlease inquire
Please inquire
1g
Please inquire
Please inquire
Please inquire
0.1 g
0.25 g0.25 g0.25 g, 0.5 g, 1 g
0.5 g
0.05 g, 0.1 g
0.5 g, 1 g
0.25 g0.25 g, 0.5 g, 1 g
0.25 g, 0.5 g
0.25 gPlease inquire
1g
Please inquire
Please inquire
0.25 g0.1 g
Please inquire
0.1 g, 0.25 g, 0.5 g
0.1 g
0.1 g, 1 g

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

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Cambridge Isotope Laboratories, Inc.

| Stable Isotopes for Biomolecular NMR

Amino Acids (continued)
Catalog No.

Description

Unit Size

NLM-557
NLM-1328
CNLM-7813
CNLM-1275-H
DNLM-6997
CDNLM-6805
CLM-422
CLM-136
CLM-1017
DLM-1674
DLM-280
DLM-280-80
NLM-202
CNLM-507
CNLM-508
CNLM-1673-H
DNLM-6862
CDNLM-6799
NLM-4649
NLM-4457
NLM-9585
CLM-1512
CLM-2264
DLM-7855
NLM-2245
NLM-846
NLM-1513
CNLM-758
DNLM-7366
CDNLM-6806
CLM-1026
CLM-2248-H
DLM-141
NLM-292
CNLM-7810
CNLM-561-H
DNLM-7325
CDNLM-6807
CLM-468
CLM-2014
CLM-3524
CLM-2262-H
DLM-1259
DLM-4212
DLM-567
NLM-142
CNLM-615
CNLM-615-95
CNLM-3450
CNLM-281-H
DNLM-4642
CDNLM-6808

L-Glutamine (amide-15N, 98%)
L-Glutamine (15N2, 98%)
L-Glutamine (1-13C, 99%; a-15N, 98%)
L-Glutamine (13C5, 99%; 15N2, 99%)
L-Glutamine (2,3,3,4,4-D5, 97-98%; 15N2, 97-98%)
L-Glutamine (13C5, 97-99%; D5, 97-99%; 15N2, 97-99%)
Glycine (1-13C, 99%)
Glycine (2-13C, 99%)
Glycine (1,2-13C2, 97-99%)
Glycine (2,2-D2, 98%)
Glycine (D5, 98%)
Glycine (D5, 80%)
Glycine (15N, 98%)
Glycine (1-13C, 99%; 15N, 98%)
Glycine (2-13C, 99%; 15N, 98%)
Glycine (13C2, 99%; 15N, 99%)
Glycine (2,2-D2, 98%; 15N, 98%)
Glycine (13C2, 97-99%; 2,2-D2, 97-99%; 15N, 97-99%)
L-Histidine (ring-e-15N, 98%)
L-Histidine (ring-p-15N, 98%)
L-Histidine (ring-15N2, 98%)
L-Histidine·HCl·H2O (ring-2-13C, 99%)
L-Histidine·HCl·H2O (13C6, 97-99%)
L-Histidine·HCl·H2O (ring-2,4-D2; a,b,b-D3, 98%)
L-Histidine·HCl·H2O (a-15N, 98%)
L-Histidine·HCl·H2O (ring-p-15N, 98%)
L-Histidine·HCl·H2O (15N3, 98%)
L-Histidine·HCl·H2O (13C6, 97-99%; 15N3, 97-99%) (<5% D)
L-Histidine·HCl·H2O (D5, 98%; 15N3, 98%)
L-Histidine·HCl·H2O (13C6, 97-99%; D5, 97-99%; 15N3, 97-99%)
L-Isoleucine (1-13C, 99%)
L-Isoleucine (13C6, 99%)
L-Isoleucine (D10, 98%)
L-Isoleucine (15N, 98%)
L-Isoleucine (1-13C, 99%; 15N, 98%)
L-Isoleucine (13C6, 99%; 15N, 99%)
L-Isoleucine (D10, 98%; 15N, 98%)
L-Isoleucine (13C6, 97-99%; D10, 97-99%; 15N, 97-99%)
L-Leucine (1-13C, 99%)
L-Leucine (2-13C, 99%)
L-Leucine (1,2-13C2, 99%)
L-Leucine (13C6, 99%)
L-Leucine (5,5,5-D3, 99%)
L-Leucine (isopropyl-D7, 98%)
L-Leucine (D10, 98%)
L-Leucine (15N, 98%)
L-Leucine (1-13C, 99%; 15N, 98%)
L-Leucine (1-13C, 99%; 15N, 93-95%)
L-Leucine (2-13C, 99%; 15N, 95-99%)
L-Leucine (13C6, 99%; 15N, 99%)
L-Leucine (D10, 98%; 15N, 97%)
L-Leucine (13C6, 97-99%; D10, 97-99%; 15N, 97-99%)

0.5 g, 1 g
0.25 gPlease inquire
0.1 g, 0.25 g, 0.5 g
0.25 g0.25 g
1 g, 5 g
0.5 g, 1 g, 5 g
0.5 g, 1 g, 5 g
5g
5g
5g
1 g, 5 g
1g
0.5 g, 1 g
0.25 g, 0.5 g, 1 g
0.25 g, 0.5 g
0.25 gPlease inquire
Please inquire
Please inquire
0.1 g
0.05 g, 0.1 g, 0.25 g
0.25 g0.25 gPlease inquire
0.25 g0.25 g0.25 g0.25 g
0.5 g, 1 g
0.05 g, 0.1 g, 0.25 g
0.1 g, 0.25 g
0.25 gPlease inquire
0.05 g, 0.1 g, 0.25 g
0.25 g0.25 g
1 g, 5 g
0.5 g, 1 g
0.25 g0.05 g, 0.1 g, 0.25 g
1 g, 5 g
1g
0.25 g
0.5 g, 1 g
1g
1g
0.5 g
0.05 g, 0.1 g, 0.25 g
0.25 g, 0.5 g
0.25 gChemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

30

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| Stable Isotopes for Biomolecular NMR

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Catalog No.

Description

Unit Size

CLM-653
CLM-632
CLM-2247-H
DLM-2640
DLM-2641
DLM-570
NLM-143
NLM-631
NLM-1554
CNLM-7821
CNLM-3454
CNLM-291-H
DNLM-7545
CDNLM-6810
ULM-8766
CLM-206
CLM-3267
CLM-893-H
DLM-431
DLM-6797
NLM-752
CDLM-760
CDLM-9289
CDLM-8885
CNLM-7807
CNLM-9774
CNLM-759-H
DNLM-7179
CDNLM-6798
CLM-1036
CLM-4724-H
DLM-4261
DLM-6046
DLM-2969
DLM-6669
NLM-2212
NLM-2174
NLM-3610
CDLM-3873
CNLM-7578-H
CLM-762
CLM-1631
CLM-1053
CLM-1055
CLM-2250-H
DLM-2984
DLM-2985
DLM-1258
DLM-372
NLM-108
CNLM-7611
CNLM-575-H

L-Lysine·2HCl (1-13C, 99%)
L-Lysine·2HCl (6-13C, 99%)
L-Lysine·2HCl (13C6, 99%)
L-Lysine·2HCl (4,4,5,5-D4, 96-98%)
L-Lysine·2HCl (3,3,4,4,5,5,6,6-D8, 98%)
L-Lysine·2HCl (D9, 98%)
L-Lysine·2HCl (a-15N, 95-99%)
L-Lysine·2HCl (e-15N, 98%)
L-Lysine·2HCl (15N2, 98%)
L-Lysine·2HCl (1-13C, 99%; a-15N, 98%)
L-Lysine·2HCl (6-13C, 99%; e-15N, 98%)
L-Lysine·2HCl (13C6, 99%; 15N2, 99%)
L-Lysine·2HCl (D9, 98%; 15N2, 98%)
L-Lysine·2HCl (13C6, 97-99%; D9, 97-99%; 15N2, 97-99%)
L-Lysine·2HCl (unlabeled)
L-Methionine (methyl-13C, 99%)
L-Methionine (1-13C, 99%)
L-Methionine (13C5, 99%)
L-Methionine (methyl-D3, 98%)
L-Methionine (2,3,3,4,4-D5; methyl-D3, 98%)
L-Methionine (15N, 96-98%)
L-Methionine (1-13C, 99%; methyl-D3, 98%)
L-Methionine (methyl-13C, 99%; methyl-D3, 98%)
L-Methionine (methyl-13CH3, 99%; 2,3,3,4,4-D5, 98%)
L-Methionine (1-13C, 99%; 15N, 98%)
L-Methionine (1,2,3,4-13C4, 99%; 15N, 98%)
L-Methionine (13C5, 99%; 15N, 99%)
L-Methionine (D8, 98%; 15N, 98%)
L-Methionine (13C5, 97-99%; D8, 97-99%; 15N, 97-99%)
L-Ornithine·HCl (1,2-13C2, 99%)
L-Ornithine·HCl (13C5, 99%)
L-Ornithine·HCl (5,5-D2, 98%)
L-Ornithine·HCl (4,4,5,5-D4, 95%)
L-Ornithine·HCl (3,3,4,4,5,5-D6, 98%)
L-Ornithine·HCl (D7, 98%)
L-Ornithine·HCl (a-15N, 98%)
L-Ornithine·HCl (5-15N, 98%)
L-Ornithine·HCl (15N2, 98%)
L-Ornithine·HCl (5-13C, 99%; 4,4,5,5-D4, 95%)
L-Ornithine·HCl (13C5, 99%; 15N2, 99%)
L-Phenylalanine (1-13C, 99%)
L-Phenylalanine (2-13C, 99%) CP 97%
L-Phenylalanine (3-13C, 99%)
L-Phenylalanine (ring-13C6, 99%)
L-Phenylalanine (13C9, 99%)
L-Phenylalanine (2-D, 95%)
L-Phenylalanine (3,3-D2, 98%)
L-Phenylalanine (ring-D5, 98%)
L-Phenylalanine (D8, 98%)
L-Phenylalanine (15N, 98%)
L-Phenylalanine (2,3-13C2, 99%; 15N, 98%)
L-Phenylalanine (13C9, 99%; 15N, 99%)

0.25 g, 0.5 g
0.25 g0.05 g, 0.1 g, 0.25 g, 0.5 g, 1 g
0.25 g, 0.5 g, 1 g
0.25 g0.1 g0.25 g, 1 g
0.5 g
0.1 g
Please inquire
Please inquire
0.05 g, 0.1 g, 0.25 g, 0.5 g, 1 g
0.25 g0.25 g0.05 g, 0.1 g
1g
0.25 g, 1 g
0.05 g, 0.1 g, 0.25 g
1 g, 5 g
0.1 g
0.5 g, 1 g
Please inquire
0.25 g, 1 g
0.5 g, 1 g
0.25 gPlease inquire
0.05 g, 0.1 g, 0.25 g
0.25 gPlease inquire
Please inquire
0.1 g
0.25 gPlease inquire
0.1 g, 0.25 g
0.25 gPlease inquire
Please inquire
0.25 gPlease inquire
Please inquire
1g
0.05 g, 0.25 g
0.1 g, 0.25 g
0.25 g0.25 g, 0.5 g, 1 g
0.5 g
0.1 g, 0.5 g, 1 g
1 g, 5 g
1g
0.5 g, 1 g
Please inquire
0.1 g, 0.25 g, 0.5 g, 1 g

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

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Cambridge Isotope Laboratories, Inc.

| Stable Isotopes for Biomolecular NMR

Amino Acids (continued)
Catalog No.

Description

Unit Size

DNLM-7180
CDNLM-6811
CLM-510
CLM-2260-H
DLM-487
NLM-835
CNLM-7822
CNLM-436-H
DNLM-7562
CDNLM-6812
CLM-1573
CLM-2013
CLM-1572
CLM- 1574-H
DLM-161
DLM-582
NLM-2036
CNLM-7814
CNLM-474-H
DNLM-6863
CDNLM-6813
CLM-447
CLM-2261
DLM-1693
NLM-742
CDLM-9307
CNLM-587
DNLM-7367
CDNLM-6814
CLM-778
CLM-1543
CLM-716
CLM-717
CLM-4290-H
DLM-1092
DLM-6903
NLM-1695
NLM-1208
NLM-800
CNLM-2475-H
DNLM-6904
CDNLM-6816
CLM-776
CLM-437
CLM-3378
CLM-622
CLM-623
CLM-1542
CLM-2263-H
DLM-2317
DLM-449
DLM-2917

L-Phenylalanine (D8, 98%; 15N, 98%)
L-Phenylalanine (13C9, 97-99%; D8, 97-99%; 15N, 97-99%)
L-Proline (1-13C, 99%)
L-Proline (13C5, 99%)
L-Proline (D7, 97-98%)
L-Proline (15N, 98%)
L-Proline (1-13C, 99%; 15N, 98%)
L-Proline (13C5, 99%; 15N, 99%)
L-Proline (D7, 98%; 15N, 98%)
L-Proline (13C5, 97-99%; D7, 97-99%; 15N, 97-99%)
L-Serine (1-13C, 99%)
L-Serine (2-13C, 99%)
L-Serine (3-13C, 99%)
L-Serine (13C3, 99%)
L-Serine (3,3-D2, 98%)
L-Serine (2,3,3-D3, 98%)
L-Serine (15N, 98%)
L-Serine (1-13C, 99%; 15N, 98%)
L-Serine (13C3, 99%; 15N, 99%)
L-Serine (2,3,3-D3, 98%; 15N, 98%)
L-Serine (13C3, 97-99%; D3, 97-99%; 15N, 97-99%)
L-Threonine (1-13C, 99%)
L-Threonine (13C4, 97-99%)
L-Threonine (D5, 98%)
L-Threonine (15N, 98%)
L-Threonine (4-13C, 97%; 2,3-D2, 98%)
L-Threonine (13C4, 97-99%; 15N, 97-99%)
L-Threonine (D5, 97%; 15N, 98%)
L-Threonine (13C4, 97-99%; D5, 97-99%; 15N, 97-99%)
L-Tryptophan (1-13C, 99%)
L-Tryptophan (indole-2-13C, 98%)
L-Tryptophan (indole-3-13C, 95-99%)
L-Tryptophan (indole-4-13C, 99%) CP 95%
L-Tryptophan (13C11, 99%)
L-Tryptophan (indole-D5, 98%)
L-Tryptophan (D8, 97-98%)
L-Tryptophan (a-15N, 95-99%)
L-Tryptophan (indole-15N, 98%)
L-Tryptophan (15N2, 98%)
L-Tryptophan (13C11, 99%; 15N2, 99%)
L-Tryptophan (D8, 98%; 15N2, 98%)
L-Tryptophan (13C11, 97-99%; D8, 97-99%; 15N2, 97-99%)
L-Tyrosine (1-13C, 99%)
L-Tyrosine (2-13C, 99%)
L-Tyrosine (3-13C, 99%)
L-Tyrosine (phenol-4-13C, 95-99%)
L-Tyrosine (phenol-3,5-13C2, 95-99%)
L-Tyrosine (ring-13C6, 99%)
L-Tyrosine (13C9, 99%)
L-Tyrosine (3,3-D2, 98%)
L-Tyrosine (ring-3,5-D2, 98%)
L-Tyrosine (ring-2,6-D2, 2-D, 98%)

0.25 g, 0.5 g
0.25 g0.25 g
0.1 g, 0.25 g, 0.5 g
0.1 g, 0.25 g
0.25 g, 0.5 g
Please inquire
0.1 g, 0.25 g, 0.5 g
0.25 g0.25 g0.25 g
0.1 g
0.1 g, 0.25 g
0.1 g, 0.25 g, 0.5 g
Please inquire
0.1 g, 0.5 g
0.5 g, 1 g
0.25 g
0.1 g, 0.25 g, 0.5 g
0.25 g0.25 g
0.5 g
0.1 g, 0.25 g, 0.5 g
Please inquire
0.25 g, 0.5 g
0.1 g, 0.5 g
0.1 g, 0.25 g, 0.5 g
0.25 g, 0.5 g
0.25 g0.25 g0.25 g0.25 gPlease inquire
0.1 g
0.5 g
0.25 g0.1 g0.25 g, 0.5 g
0.25 g, 0.5 g
0.1 g
0.25 g0.25 g1g
0.1 g, 0.25 g
0.1 g, 0.25 g
0.25 g0.25 g0.25 g
0.1 g, 0.25 g, 0.5 g
0.5 g, 1 g
1 g, 5 g
Please inquire

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

32

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| Stable Isotopes for Biomolecular NMR

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Catalog No.

Description

Unit Size

DLM-451
DLM-589
NLM-590
CDLM-2369
CNLM-7809
CNLM-7610
CNLM-439-H
DNLM-7373
NOLM-10743
CDNLM-6815
CLM-470
CLM-3050
CLM-9217
CLM-2249-H
DLM-7732
DLM-4364
DLM-488
NLM-316
CNLM-3466
CNLM-8678
CNLM-442-H
DNLM-4643
NOLM-10742
CDNLM-4281
CDNLM-6817

L-Tyrosine (ring-D4, 98%)
L-Tyrosine (D7, 98%)
L-Tyrosine (15N, 98%)
L-Tyrosine (ring-13C6, 99%; 3,3-D2, 30%)
L-Tyrosine (1-13C, 99%; 15N, 98%)
L-Tyrosine (2,3-13C2, 99%; 15N, 98%)
L-Tyrosine (13C9, 99%; 15N, 99%)
L-Tyrosine (D7, 98%; 15N, 98%)
L-Tyrosine (15N, 98%; carboxyl-17O2, 60%)
L-Tyrosine (13C9, 97-99%; D7, 97-99%; 15N, 97-99%)
L-Valine (1-13C, 99%)
L-Valine (2-13C, 99%)
L-Valine (dimethyl-13C2, 99%)
L-Valine (13C5, 99%)
L-Valine (3-D, 98%)
L-Valine (2,3-D2, 98%)
L-Valine (D8, 98%)
L-Valine (15N, 98%)
L-Valine (1-13C, 99%; 15N, 98%)
L-Valine (2-13C, 99%; 15N, 98%)
L-Valine (13C5, 99%; 15N, 99%)
L-Valine (D8, 96%; 15N, 96%)
L-Valine (15N, 98%; carboxyl-17O2, 60%)
L-Valine (13C5, 95-97%; 2,3-D2, 97%; 15N, 96-99%)
L-Valine (13C5, 97-99%; D8, 97-99%; 15N, 97-99%)

0.5 g, 1 g
Please inquire
0.5 g
0.1 g
0.25 gPlease inquire
0.1 g, 0.25 g, 0.5 g
0.25 gPlease inquire
0.25 g1g
0.25 g0.25 g, 1 g
0.25 g, 1 g, 5 g
1g
0.1 g, 0.25 g
0.25 g, 0.5 g
0.5 g, 1 g
0.25 gPlease inquire
0.25 g, 0.5 g, 1 g
0.25 g, 0.5 g
Please inquire
0.1 g, 0.25 g
0.25 gChemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

cilsales@isotope.com | intlsales@isotope.com | isotope.com 33

Cambridge Isotope Laboratories, Inc.

| Stable Isotopes for Biomolecular NMR

Protected Amino Acids
CIL offers a comprehensive list of labeled protected amino acids for use as starting materials for peptide synthesis. The large number ofavailable labeling schemes maximizes flexibility involved in designing experiments to probe structure and dynamics alone or in the presenceof biological macromolecules. Pricing and availability can be found on isotope.com.
Catalog No.
CLM-818
CLM-3638
CLM-1142
CLM-7785
DLM-7316
DLM-8168
NLM-614
CNLM-4355-H
CDNLM-7852
CLM-2150
CLM-2011
CLM-2151
CLM-3589
DLM-649
DLM-2793
NLM-1903
CNLM-6014
CNLM-2394
CLM-8475-H
NLM-8841
CNLM-8474-H
NLM-1264
CNLM-4354
CNLM-6193-H
NLM-4204
CNLM-4788
NLM-647
NLM-3493
NLM-1908
CNLM-2392
DLM-4721
CNLM-4722-H
CLM-1901
NLM-3874
NLM-8960
CNLM-4753-H
CLM-2008
NLM-1907
CLM-1902
NLM-3419
CLM-3639
CLM-2387
CLM-7547
DLM-7339
NLM-694
CNLM-7697
CNLM-7698
CNLM-4357-H
CDNLM-7853

Description
L-Alanine-N-FMOC (1-13C, 99%)
L-Alanine-N-FMOC (2-13C, 99%)
L-Alanine-N-FMOC (3-13C, 99%)
L-Alanine-N-FMOC (13C3, 97-99%)
L-Alanine-N-FMOC (3,3,3-D3, 98%)
L-Alanine-N-FMOC (2,3,3,3-D4, 98%)
L-Alanine-N-FMOC (15N, 98%)
L-Alanine-N-FMOC (13C3, 99%; 15N, 99%)
L-Alanine-N-FMOC (13C3, 97-99%; D4, 97-99%; 15N, 97-99%)
L-Alanine-N-t-BOC (1-13C, 99%)
L-Alanine-N-t-BOC (2-13C, 98-99%)
L-Alanine-N-t-BOC (3-13C, 99%)
L-Alanine-N-t-BOC (13C3, 97-99%)
L-Alanine-N-t-BOC (2-D, 98%)
L-Alanine-N-t-BOC (3,3,3-D3, 99%)
L-Alanine-N-t-BOC (15N, 98%)
L-Alanine-N-t-BOC (2-13C, 99%; 15N, 96-99%)
L-Alanine-N-t-BOC (13C3, 97-99%; 15N, 97-99%)
L-Arginine-N-FMOC, PBF-OH (13C6, 99%) contains solvent
L-Arginine-N-FMOC, PBF-OH (15N4, 98%) contains solvent
L-Arginine-N-FMOC, PBF-OH (13C6, 99%; 15N4, 99%) contains solvent
L-Arginine-N-FMOC, PMC (15N4, 98%) CP 94%
L-Asparagine-N-FMOC (13C4, 97-99%; 15N2, 97-99%)
L-Asparagine-N-FMOC, N-b-trityl (13C4, 99%; 15N2, 99%)
L-Asparagine-N-FMOC, N-b-trityl (15N2, 98%)
L-Aspartic acid-N-FMOC (13C4, 97-99%; 15N, 97-99%)
L-Aspartic acid-N-FMOC, b-O-t-butyl ester (15N, 98%)
L-Aspartic acid-N-t-BOC (15N, 98%)
L-Aspartic acid-N-t-BOC, b-Bz ester (15N, 98%)
L-Aspartic acid-N-t-BOC, b-Bz ester (13C4, 97-99%; 15N, 97-99%)
L-Cysteine-N-FMOC, S-trityl (3,3-D2, 98%)
L-Cysteine-N-FMOC, S-trityl (13C3, 99%; 15N, 99%)
L-Cysteine-N-t-BOC, S-benzyl (3-13C, 99%)
L-Cysteine-N-t-BOC, S-P-mebz (15N, 98%)
L-Glutamic acid-N-FMOC, g-t-butyl ester (15N, 98%)
L-Glutamic acid-N-FMOC, g-t-butyl ester (13C5, 99%; 15N, 99%) CP 96%
L-Glutamic acid-N-t-BOC, g-benzyl ester (1,2-13C2, 99%)
L-Glutamic acid-N-t-BOC, g-benzyl ester (15N, 98%)
L-Glutamine-N-t-BOC (1,2-13C2, 99%)
L-Glutamine-N-t-BOC (a-15N, 98%)
Glycine-N-FMOC (1-13C, 99%)
Glycine-N-FMOC (2-13C, 99%)
Glycine-N-FMOC (13C2, 97-99%)
Glycine-N-FMOC (2,2-D2, 98%)
Glycine-N-FMOC (15N, 98%)
Glycine-N-FMOC (1-13C, 99%; 15N, 98%)
Glycine-N-FMOC (2-13C, 99%; 15N, 98%)
Glycine-N-FMOC (13C2, 99%; 15N, 99%)
Glycine-N-FMOC (13C2, 97-99%; 2,2-D2, 97-99%; 15N, 97-99%)

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

34

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Unit Size
1g
0.25 g1g
0.25 g1g
0.5 g
1g
0.1 g, 0.25 g
Please inquire
1g
0.25 g
0.5 g, 1 g
0.25 gPlease inquire
1g
0.25 g, 0.5 g, 1 g
Please inquire
0.05 g, 0.1 g
1g
Please inquire
0.1 g, 0.25 g, 0.5 g, 1 g
0.1 g
Please inquire
0.1 g
0.1 g
0.05 g
0.1 g, 0.5 g, 1 g
1g0.25 g0.05 g
0.1 g, 0.25 g, 0.5 g
0.1 g, 0.25 g
0.25 g0.25 g
0.1 g
0.1 g
0.1 g
Please inquire
0.1 g
0.5 g
1g
1g
1g
1g
1g
0.25 g
0.1 g
0.1 g, 0.25 g
Please inquire| Stable Isotopes for Biomolecular NMR

isotope.com

Catalog No.

Description

Unit Size

CLM-2152
CLM-1900
DLM-2153
NLM-2109
CNLM-9686
CNLM-2412
NLM-8010
CLM-8043
CLM-7794
NLM-391
NLM-2167
CLM-3691
CLM-7546
CLM-3683
DLM-7202
DLM-7575
NLM-2397
CNLM-4345-H
CDNLM-7854
CLM-2155
CLM-2010
DLM-2736
DLM-3650
NLM-1904
CNLM-2396
CLM-6194
CLM-7865-H
NLM-4631
CNLM-4754-H
CLM-8166
CLM-1141
NLM-4632
CNLM-4358-H
CLM-2156
CLM-4824
CLM-3684
DLM-7786
DLM-8752
NLM-1265
CNLM-4362-H
CLM-2170
CLM-2009
CLM-2061
CLM-7859
DLM-2157
NLM-1905
CNLM-2393
CLM-8044
NLM-3906
NLM-2329
CNLM-4347-H

Glycine-N-t-BOC (1-13C, 99%)
Glycine-N-t-BOC (2-13C, 99%)
Glycine-N-t-BOC (2,2-D2, 98%)
Glycine-N-t-BOC (15N, 98%)
Glycine-N-t-BOC (2-13C, 99%; 15N, 98%)
Glycine-N-t-BOC (13C2, 97-99%; 15N, 97-99%)
L-Histidine-N-FMOC, N-Im-trityl (15N3, 98%)
L-Isoleucine-N-FMOC (1-13C, 99%)
L-Isoleucine-N-FMOC (13C6, 97-99%)
L-Isoleucine-N-FMOC (15N, 98%)
L-Isoleucine-N-t-BOC (15N, 98%)
L-Leucine-N-FMOC (1-13C, 99%)
L-Leucine-N-FMOC (1,2-13C2, 99%)
L-Leucine-N-FMOC (13C6, 97-99%)
L-Leucine-N-FMOC (5,5,5-D3, 98%)
L-Leucine-N-FMOC (D10, 98%)
L-Leucine-N-FMOC (15N, 98%)
L-Leucine-N-FMOC (13C6, 99%; 15N, 99%)
L-Leucine-N-FMOC (13C6, 97-99%; D10, 97-99%; 15N, 97-99%)
L-Leucine-N-t-BOC·H2O (1-13C, 99%)
L-Leucine-N-t-BOC·H2O (2-13C, 99%)
L-Leucine-N-t-BOC·H2O (5,5,5-D3, 98%)
L-Leucine-N-t-BOC·H2O (D10, 98%)
L-Leucine-N-t-BOC·H2O (15N, 98%)
L-Leucine-N-t-BOC·H2O (13C6, 97-99%; 15N, 97-99%)
L-Lysine-a-N-FMOC, e-N-t-BOC (1-13C, 99%)
L-Lysine-a-N-FMOC, e-N-t-BOC (13C6, 99%)
L-Lysine-a-N-FMOC, e-N-t-BOC (15N2, 96-98%)
L-Lysine-a-N-FMOC, e-N-t-BOC (13C6, 99%; 15N2, 99%)
L-Methionine-N-FMOC (1-13C, 99%)
L-Methionine-N-FMOC (methyl-13C, 99%)
L-Methionine-N-FMOC (15N, 98%) CP 95%
L-Methionine-N-FMOC (13C5, 97-99%; 15N, 97-99%)
L-Methionine-N-t-BOC (methyl-13C, 98%)
L-Phenylalanine-N-FMOC (1-13C, 99%)
L-Phenylalanine-N-FMOC (ring-13C6, 99%)
L-Phenylalanine-N-FMOC (ring-D5, 98%)
L-Phenylalanine-N-FMOC (D8, 98%)
L-Phenylalanine-N-FMOC (15N, 98%)
L-Phenylalanine-N-FMOC (13C9, 99%; 15N, 99%)
L-Phenylalanine-N-t-BOC (1-13C, 99%)
L-Phenylalanine-N-t-BOC (2-13C, 99%)
L-Phenylalanine-N-t-BOC (ring-13C6, 99%)
L-Phenylalanine-N-t-BOC (13C9, 97-99%)
L-Phenylalanine-N-t-BOC (ring-D5, 98%)
L-Phenylalanine-N-t-BOC (15N, 98%)
L-Phenylalanine-N-t-BOC (13C9, 97-99%; 15N, 97-99%)
L-Proline-N-FMOC (1-13C, 99%)
L-Proline-N-FMOC (15N, 98%)
L-Proline-N-t-BOC (15N, 96%)
L-Proline-N-FMOC (13C5, 99%; 15N, 97-99%)

1g
1g
1g
1g
Please inquire
0.1 g
0.1 g
0.25 gPlease inquire
0.25 g0.25 g
1g
0.1 g
0.1 g
1g
0.25 g1g
0.1 g, 0.25 g
Please inquire
1g
0.25 g1g
0.5 g
1g
0.05 g0.1 g
Please inquire
0.1 g
0.1 g, 0.25 g, 0.5 g, 1 g
Please inquire
Please inquire
Please inquire
0.1 g
Please inquire
0.5 g
0.1 g
0.25 gPlease inquire1g
0.1 g, 0.25 g
0.5 g
0.25 g0.1 g0.05 g
1g
1g
0.05 g0.25 g0.25 g0.25 g
0.1 g, 0.25 g

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

cilsales@isotope.com | intlsales@isotope.com | isotope.com 35

Cambridge Isotope Laboratories, Inc.

| Stable Isotopes for Biomolecular NMR

Protected Amino Acids (continued)
Cata log No.

Description

Unit Size

CNLM-8403-H
CLM-8167
NLM-4630
CNLM-4755-H
CLM-2007
CLM-756
NLM-2025
NLM-8170
CNLM-7615-H
NLM-3681
DLM-6113
NLM-3423
CNLM-6077
CLM-2194
CNLM-4349-H
NLM-8169
DLM-2303
NLM-1906
CLM-3640
CLM-7793
DLM-7784
NLM-4243
CNLM-4348-H
CLM-2158
DLM-3651
NLM-2060
CNLM-2395

L-Serine-N-FMOC (13C3, 99%; 15N, 99%)
L-Serine-N-FMOC, O-t-butyl ether (1-13C, 99%)
L-Serine-N-FMOC, O-t-butyl ether (15N, 98%)
L-Serine-N-FMOC, O-t-butyl ether (13C3, 99%; 15N, 99%)
L-Serine-N-t-BOC, O-Bz ether (2-13C, 99%)
L-Serine-N-t-BOC, O-Bz ether (3-13C, 99%)
L-Serine-N-t-BOC, O-Bz ether (15N, 98%)
L-Threonine-N-FMOC, O-t-butyl ether (15N, 98%)
L-Threonine-N-FMOC, O-t-butyl ether (13C4, 99%; 15N, 99%)
L-Threonine-N-t-BOC, O-benzyl ether (15N, 98%)
L-Tryptophan-N-FMOC (indole-D5, 98%)
L-Tryptophan-N-FMOC (a-15N, 98%)
L-Tryptophan-N-FMOC (13C11, 97-99%; 15N2, 97-99%)
L-Tryptophan-N-t-BOC (1-13C, 99%)
L-Tyrosine-N-FMOC, O-t-butyl ether (13C9, 99%; 15N, 99%)
L-Tyrosine-N-FMOC, O-t-butyl ether (15N, 98%)
L-Tyrosine-N-t-BOC, O-Bz ether (ring-D4, 98%)
L-Tyrosine-N-t-BOC, O-Bz ether (15N, 98%)
L-Valine-N-FMOC (1-13C, 99%)
L-Valine-N-FMOC (13C5, 97-99%)
L-Valine-N-FMOC (D8, 98%)
L-Valine-N-FMOC (15N, 98%)
L-Valine-N-FMOC (13C5, 99%; 15N, 99%)
L-Valine-N-t-BOC (1-13C, 99%)
L-Valine-N-t-BOC (D8, 98%)
L-Valine-N-t-BOC (15N, 98%)
L-Valine-N-t-BOC (13C5, 97-99%; 15N, 97-99%)1g
0.25 g0.25 g0.1 g
Please inquire
0.1 g
0.1 g
0.1 g
0.1 g
Please inquire
0.25 gPlease inquire
0.1 g
Please inquire
0.1 g, 0.25 g
0.1 g
0.25 g0.25 g
1g
0.1 g
0.5 g
1g
0.1 g, 0.25 g
Please inquire
0.5 g
0.5 g, 1 g
0.05 g❛❛Over the past two decades, companies providing specificallylabeled isotopes and precursors have changed our ways andunderstanding of biomolecular NMR sample preparations. CIL,
being the widely popular company, certainly plays a crucialrole in this development. I enjoy working with CIL because of
the value, quality of products and superb service. I have beenordering from them for the past 15+ years: from Munich asa PhD student, from San Francisco as post-doc, and from
Hyderabad as a PI. To tell you the truth, I do not want toeven imagine an NMR world without isotopes and CIL.❜❜
Mandar V. Deshmukh, PhD
Scientist, Structural Biology (NMR)
Centre for Cellular and Molecular Biology

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

36

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| Stable Isotopes for Biomolecular NMR

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Sparse Labeling Reagents
The presence of one bond 13C-13C coupling in proteins may present
difficulties in obtaining structural information in both solid-stateand solution NMR. It is easier to overcome these difficulties bysimply analyzing sparsely 13C-labeled protein than resorting to using
specialized pulse sequences. Sparsely labeled protein is made byexpressing protein in E. coli grown using selectively 13C-labeled
carbon sources. The use of 2-13C and 1,3-13C2 glycerol, as originallydescribed by LeMaster,1 remains to be the most popular sparselabeling method in current use. Proteins expressed using 1-13C
glucose and 2-13C glucose as sole carbon sources, however, havebeen used to determine intermolecular distances in supramolecularcomplexes using 13C-13C solid state NMR.2 CIL offers the following
selectively 13C-labeled E. coli carbon sources.

Sparse Labeling Reagents
Catalog No.
CLM-420
CLM-746
CLM-1396-25-1
CLM-1397
CLM-1857
CLM-1580
CLM-1575

Description
D-Glucose (1-13C, 99%)
D-Glucose (2-13C, 98-99%)
D-Glucose (13C6, 24-25%)
Glycerol (2-13C, 99%)
Glycerol (1,3-13C2, 99%)
Sodium pyruvate (2-13C, 99%)
Sodium pyruvate (3-13C, 99%)

References
1.	Wylie, B.J.; Schwieters, C.D.; Oldfield, E.; et al. 2009. Protein structure refinement
using 13Cα chemical shift tensors. J Am Chem Soc, 131(3), 985-992.
2.	Loquet, A.; Giller, K.; Becker, S.; et al. 2010. Supramolecular interactions probed
by 13C-13C solid-state NMR spectroscopy. J Am Chem Soc, 132, 15164-15166.

Mixed Pyruvate Labeling
A backbone resonance assignment strategy was recently published1
which describes the use of an equimolar mix of 2-13C and 3-13C
sodium pyruvate as the sole carbon source for the expressionof recombinant 13C sparsely labeled protein in E. coli cells.
The achieved sparse 13C labeling suppresses one-bond (1Jab)
coupling, which provides enhanced resolution for the Caresonance and results in distinct peaks shapes for each aminoacid in 1D traces along the Ca dimension of 3D HNCA spectra.
This method allows the near total assignment of backbone
resonances of large proteins using a single HNCA experiment,
reducing the required spectrometer time compared to current
resonance assignment procedures.

Pyruvate 13C Labeling Mix
Catalog No.
CLM-10978-KIT
NEW!

Description
1 vial containing 2-13C sodium pyruvate (1.5 g)(CLM-1580)
1 vial containing 3-13C sodium pyruvate (1.5 g)(CLM-1575)

References
1.	Robson, S.A.; Takeuchi, K.; Boeszoermenyi, A.; et al. 2018. Mixed pyruvate labelingenables backbone resonance assignment of large proteins using a single experiment.
Nat Commun, 9(1), 356.

CIL is pleased to assist researchers using this technique by suppying
prepackaged amounts of 2-13C and 3-13C sodium pyruvate in abundled packaged, referred to as “Hari’s Mix.”

Recommended Preparation of Growth Media with Deuterated Mixed Pyruvates*
Step 1. In a suitable container, dissolve contents of both vials in 1 kg of D2O (99.9%) (DLM-4).
Step 2.	Adjust pH to ~13 by adding 2.5 mM NaOD (prepared using 40% NaOD in D2O – DLM-45), while stirring. Wait 30
minutes, stirring occassionally.
Step 3. Add 4.26 g Na2HPO4, 3.6 g NaH2PO4, and 3 g KH2PO4 to reach pH 7.
Step 4.	Add remaining compounds to medium. These ingredients will generally include ammonium chloride, sodium
bicarbonate, magnesium sulfate, calcium chloride, and antibiotics.
Step 5. Sterile filter the medium prior to use.
*This medium is not compatible with rich media additives, such as Celtone or BioExpress 1000.

cilsales@isotope.com | intlsales@isotope.com | isotope.com 37

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Deuterated Detergents and Phospholipids for Membrane Proteins
Membrane proteins can be divided into three categories:
1.	Integral membrane proteins, which penetrate the lipid
bilayer;
2.	Peripheral membrane proteins, which are externaland bound through noncovalent interactions; and3.	Lipid-anchored proteins, which are external, but boundwith covalent bonds.

Deuterated Detergents and Phospholipids

There is great interest in determining structure of integral
membrane proteins due to the importance of these proteinsin participating in cellular processes. Despite the significant
functional importance of membrane proteins, the structural
biology has been particularly challenging.

DLM-6726

N-Octyl β-Glucoside (D24, 98%)

DLM-4341

DL-A-Phosphatidylcholine, dihexanoyl (D40, 98%) (DHPC)
CP 95%

DLM-8256

DL-A-Phosphatidylcholine, dipalmitoyl (DPPC) (D80, 98%) CP
95%+

DLM-197

Sodium dodecyl sulfate (D25, 98%)

The determination of structure and dynamics of membraneproteins using NMR requires samples containing proteinthat is properly folded. Fortunately, membrane proteins often
keep native-like structures in detergent micelles. Deuterated
solubilization agents such as detergents often make NMR
investigations easier compared to using unlabeled agents.
In some cases, as in methyl labeling, deuterated reagentsof this type are required. CIL is pleased to offer deuterated
detergents and phospholipid agents for use with membraneproteins.

❛❛The methodological development that NMR has seen in thepast six decades would not have been possible without close
collaborations with companies that love to see progress
happening. CIL is a particularly strong and supportive partner,
and many ideas with respect to labeling patterns that looked
crazy on the first sight became reality through CIL, enabling
us to conquer new areas for NMR structure determination.
There is a wide variety of examples where talking to CIL has
pushed science forward.❜❜Hartmut Oschkinat, PhD
Department of NMR – Supported Structural Biology
FMP Berlin38

tel: +1.978.749.8000 | 1.800.322.1174 (USA) | fax: +1.978.749.2768

Catalog No. Description
DLM-2274
Dodecylphosphocholine (D38, 98%)

| Stable Isotopes for Biomolecular NMR

isotope.com

The Magic of MAS DNP
Robert Guy Griffin, PhD; Kong Ooi Tan, PhD; Brian C. MichaelFrancis Bitter Magnet Laboratory, Massachusetts Institute of Technology, Cambridge, MA USA

Investigator
Spotlight

Dynamic nuclear polarization (DNP)1 is a hyperpolarization
technique that enhances the sensitivity of NMR by transferring
polarization from electrons to nuclei with a theoretical maximum
enhancement factor of up to 658 when the nucleus is 1H. In a DNP
sample, paramagnetic species such as stable organic radicals2-5 aredispersed into a glassy matrix containing the sample of interest,
which is then studied in magic angle spinning (MAS) probes
capable of operating at cryogenic temperatures (< 110 K). The
key instrumentation that enables MAS-DNP at high fields is the
gyrotron, which provides the high-power microwave irradiationneeded for DNP experiments. The use of gyrotrons for DNP wasdeveloped here at MIT and later made commercially available
from Bruker.6,7
Sensitivity enhancements from DNP have allowed us to study
important biological samples such as amyloid fibrils8 and membraneproteins9 with NMR experiments that are otherwise not feasible
within a reasonable timeframe. For instance, Figure 1 showsa spectrum of 13C/ 15N-labeled urea with a ~400-fold signal
enhancement from DNP.10 In other words, the DNP-enhanced
experiment performed in an hour would have taken ~18 years
without DNP to obtain the spectrum with the same signal-tonoise ratio.
In order to remain intact under the conditions used in DNP
experiments, biological samples usually need to be cryoprotected
in a matrix that forms a glass at low temperatures. This matrix istypically a 6:3:1 mixture of d8-glycerol, D2O, and H2O, which isuniversally known as “DNP juice.” The matrix can be further
optimized by using deuterated 13C-depleted glycerol tosuppress unwanted 13C background signals. Additionally, partial
deuteration of biological samples has been shown to improve DNP
enhancements.11 2H/ 13C/ 15N-labeled samples can be prepared byexpressing proteins and peptides in E. coli in a D2O-based medium
using 15N-ammonium chloride, and 2H/ 13C-glucose. We have also
made use of a variety of sparsely labeled samples prepared using
selectively 13C-labeled 1,6-13C2-glucose or 2-13C- and 1,3-13C2glycerol.
DNP has also been applied to a variety of other problems, suchas pharmaceutical products12 and inorganic materials.13 While
MAS-NMR studies make extensive use of 1H, 15N, 13C, and even 31P,
very little work has been done on 17O despite its high abundancein biological systems. Historically, 17O studies have been limited,
because the NMR-active isotope, 17O, has a large quadrupolar
coupling and low sensitivity. We are currently developing 17O-based
experiments for use at higher magnetic fields and in conjunction
with improved sensitivity from DNP.14,15 We foresee continued
development of 17O spectroscopy which should enable theextraction of rich information from biological samples in the
future.

Figure 1. MAS-DNP on 13C/ 15N urea in “DNP juice.”
References
1.	Abragam, A; Goldman, M.1978. Principles of dynamic nuclear polarisation. Reports
Prog Phys, 41(3), 395-467.
2.	Song, C.; Hu, K.-N.; Joo, C.-G.; et al. 2006. TOTAPOL: a biradical polarizing agent
for dynamic nuclear polarization experiments in aqueous media. J Am Chem Soc,
128(35), 11385-11390.
3.	Sauvée, C.; Rosay, M.; Casano, G.; et al. 2013. Highly efficient, water-soluble
polarizing agents for dynamic nuclear polarization at high frequency. Angew ChemInt Ed Engl, 52(41), 10858-10861.
4.	Mathies, G.; Caporini, M.A.; Michaelis, V.L.; et al. 2015. Efficient dynamic nuclear
polarization at 800 MHz /527 GHz with trityl-nitroxide biradicals. Angew Chem Int
Ed Engl, 54(40), 11770-11774.
5.	Zagdoun, A.; Casano, G.; Ouari, O.; et al. 2013. Large molecular weight nitroxide
biradicals providing efficient dynamic nuclear polarization at temperatures up to
200 K. J Am Chem Soc, 135(34), 12790-12797.
6.	Becerra, L.R.; Gerfen, G.J.; Tempkin, R.A.; et al. 1993. Dynamic nuclear polarization
with a cyclotron resonance maser at 5 T. Phys Rev Lett, 71(21), 3561-3564.
7.	Rosay, M.; Blank, M.; Engelke, F. 2016. Instrumentation for solid-state dynamic
nuclear polarization with magic angle spinning NMR. J Magn Reson, 264, 88-98.
8.	Debelouchina, G.T.; Bayro, M.J.; Fitzpatrick, A.W.; et al. 2013. Higher order amyloid
fibril structure by MAS NMR and DNP spectroscopy. J Am Chem Soc, 135(51),
19237-19247.
9.	Ni, Q.Z.; Can, T.V.; Daviso, E.; et al. 2018. Primary transfer step in the light-driven
ion pump bacteriorhodopsin: an irreversible U-turn revealed by dynamic nuclear
polarization-enhanced magic angle spinning NMR. J Am Chem Soc, 140(11),4085-4091.
10.	Can, T.V.; Ni, Q.Z.; Griffin, R.G. 2015. Mechanisms of dynamic nuclear polarization
in insulating solids. J Magn Reson, 253, 23-35.
11.	Akbey, Ü.; Franks, W.T.; Linden, A.; et al. 2010. Dynamic nuclear polarization of
deuterated proteins. Angew Chem Int Ed Engl, 49(42), 7803-7806.
12.	Ni, Q.Z.; Yang, F.; Can, T.V.; et al. 2017. In situ characterization of pharmaceuticalformulations by dynamic nuclear polarization enhanced MAS NMR. J Phys Chem B,
121(34), 8132-8141.
13.	Berruyer, P.; Emsley, L.; Lesage, A. 2018. DNP in materials science: touching thesurface. eMagRes, 7(4), 93-104.
14.	Keeler, E.G.; Michaelis, V.K.; Colvin, M.T.; et al. 2017. 17O MAS NMR correlation
spectroscopy at high magnetic fields J Am Chem Soc, 139(49), 17953-17963.
15.	Keeler, E.G.; Michaelis, V.K.; Wilson, C.B.; et al. 2019. High-resolution 17O NMR
spectroscopy of structural water. J Phys Chem B, 123(14), 3061-3067.

cilsales@isotope.com | intlsales@isotope.com | isotope.com 39

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

O Solid State NMR Spectroscopy: Coming of Age

17

Timothy A. Cross, PhD
NMR and MRI User Program Director, National High Field Magnetic Lab
Chemistry and Biochemistry Professor, Florida State University, Tallahassee, Florida USA

Investigator
Spotlight

High fields bring added sensitivity and spectral dispersion for NMR,
but the influence of high fields is much greater for quadrupolar
nuclei than for our commonly studied spin ½ nuclei.1 At the
National High Magnetic Field Lab we have recently commissioned
a powered magnet for NMR operating at 35.2 Tesla or 1500 MHz.2
As a powered magnet it has to be ramped to field (~25 min)when we need it and ramped down as soon as an experiment iscompleted. We have incorporated 17O-labeled glycine and leucine
into the antibiotic, gramicidin A (gA), a 15-amino-acid peptidethat as a dimer forms a monovalent cation selective channel.3,4The structure is an alternating sequence of D and L amino acids
that as a b-strand wraps up into a helix of 6.3 residues per turn
having a pore that supports a single file column of eight watermolecules.3,5 These waters are hydrogen bonded together forming
what is known as a water wire (Figure 1) in a space-filling view
inside the pore formed by the gA dimer that spans cellular
membranes.
The 17O solid state NMR (ssNMR) spectra are dramatically enhanced
at higher magnetic fields both in sensitivity and in linewidth.6
Here, at 35.2 T the reduction in the 17O linewidth of oriented
sample (OS) ssNMR resonances is such that 1H decoupling makes
a further dramatic difference (Figure 1) leading to the observationof a pair of resonances for a single site-labeled sample of gA,7
something we did not observe when working with spectra at 19.4
and 21.1 T.8 The 17O studies in superconducting failed to observea resolvable difference in the spectra of single site 17O-labeled gA.
In hindsight there was an asymmetry in the resonance, but we
did not anticipate that the sites in the two monomers could be
distinguished after all the high-resolution 15N OS ssNMR spectraof the gA backbone showed no evidence of two resonances whena single site was labeled.2,4 With 1H decoupling and exceptional
sensitivity the 17O spectroscopy at this field strength provided a toolthat is known to be exceptionally sensitive to hydrogen bonding.9-11
It became clear that since the structure of the two monomers wereidentical the only possible distinction between the two monomers
was the hydrogen bonding from the waters to the carbonyl oxygens
lining the pore of the channel.7

With 1H decoupling and exceptional sensitivity the 17O
spectroscopy at this field strength provided a tool thatis known to be exceptionally sensitive to hydrogenbonding.

40

tel: +1.978.749.8000 | 1.800.322.1174 (USA) | fax: +1.978.749.2768

Figure 1. The 17O OS NMR spectra of Leu10-labeled gA in liquid crystalline lipid bilayers
as a function of field strength. Note that the carbonyl oxygen labeled Leu10 (dark red) ishydrogen bonded to a water proton (white spheres), but Leu10’ in the second monomer
is not hydrogen bonded to a water proton, generating a 15 ppm chemical shiftdifference for the two resonances.7

Between the two Leu10 residues are 24 carbonyl oxygens and there
are just eight water molecules that have two protons each, but oneof the protons is required in forming the water wire, as a result
there are only eight protons that could potentially hydrogen bondto a selection of the 24 carbonyl oxygens. The water wire meansthat the waters are all oriented in the same direction from one sideof the membrane to the other, but the gA monomers are oriented
in an antiparallel fashion with both of their amino termini at thecenter of the bilayer. Consequently, the interactions of the waters
in one monomer are different from the interactions of the waters
with the other monomer. This results in a unique pair of resonancesfor each site within the water wire region of the pore.7
In addition to these 17O OS ssNMR experiments, Griffin andcoworkers have been working on the development of 17O magic
angle spinning ssNMR spectroscopy at 35.2 T12,13 – those interested
in high-field spectroscopy should get in touch with the NMR/MRIUser program at the NHMFL to apply for access to the 35.2 T
spectrometer available without any user fees, thanks to NSF.

| Stable Isotopes for Biomolecular NMR

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O Solid State NMR Spectroscopy: Coming of Age (continued)

17

References
1.	Gan, Z.; Gor’kov, P.; Cross, T.A.; et al. 2002. Seeking for higher resolution andsensitivity for NMR of quadrupolar nuclei at ultra high magnetic fields.
J Am Chem Soc, 124(20), 5634-5635.

8.	Hu, J.; Chekmenev, E.; Gan, Z.; et al. 2005. Ion solvation by channel carbonyls
characterized by 17O solid state NMR at 21 T. J Am Chem Soc, 127(34),11922-11923.

2.	Gan, Z.; Hung, I.; Wang, X.; et al. 2017. NMR spectroscopy up to 35.2 T using
a series-connected hybrid magnet. J Mag Reson, 284, 125-136.

9.	Lu, J.; Hung, I.; Brinkman, A., et al. Solid state 17O NMR reveals hydrogen bonding
energetics: not all low-barrier hydrogen bonds are strong. Angew Chem Int Ed
Engl, 56(22), 6166-6170.

3.	Ketchem, R.R.; Hu, W.; Cross, T.A. 1993. High resolution conformation ofgramicidin A in a lipid bilayer by solid-state NMR. Science, 261(5127), 1457-1460.
4.	Tian, F.; Cross, T.A. 1999. Cation transport: an example of structural based
selectivity. J Mol Biol, 285(5), 1993-2003.
5.	Pomes, R.; Roux, B. 2002. Molecular mechanism of H+ conduction in the single-filewater chain of the gramicidin channel. Biophys J, 82(5), 2304-2316.
6.	Zhu, J.; Ye, E.; Terskikh, V.; et al. 2010. Solid-state 17O NMR spectroscopy oflarge protein–ligand complexes. Angew Chem Int Ed Engl, 49(45), 8399-8402.
7.	Paulino, J.; Yi, M.; Hung, I.; et al. Functional stability of water wire-carbonyl
interactions in an ion channel. Submitted for publication.

10.	Wong, A.; Pike, K.J.; Jenkins, R.; et al. 2006. Experimental and theoretical 17O NMR
study of the influence of hydrogen bonding on C=O and O-H oxygens in carboxylic
acids. J Phys Chem A, 110(5), 1824-1835.
11.	Tang, A.W.; Kong, X.; Terskikh, V.; et al. 2016. Solid-state 17O NMR of unstableacyl-enzyme intermediates: a direct probe of hydrogen bonding interactionsin the oxyanion hole of serine proteases. J Phys Chem B, 120(43), 11142-11150.
12.	Keeler, E.G.; Michaelis, V.K.; Griffin, R.G. 2016. 17O NMR investigation of water
structure and dynamics. J Phys Chem B, 120(32), 7851-7858.
13.	Keeler, E.G.; Michaelis, V.K.; Griffin, R.G.; et al. 2019. High-resolution 17O NMR
spectroscopy of structural water. J Phys Chem B, 123(14), 3061-3067.

O NMR Reagents

17

Even though oxygen is ubiquitous to all lifeforms, the use of 17O
NMR to study biological molecules is rare, due to the low naturalabundance of the 17O isotope (e.g., 0.037%) and nonfavorable
NMR properties of the 17O nucleus (low sensitivity and very broadresonances). Thus, molecules generally require enrichment using
17
O-containing starting materials (e.g., H217O) for analysis using
17
O NMR. Despite these constraints, 17O NMR encompasses
a wide chemical shift range (~1650 ppm) and therefore holds
much promise in gaining new insight in determining structure
and the chemical behavior of biological molecules.
There has been much progress in utilizing 17O NMR to study
biological molecules. For example, the laboratory of Dr. Gang
Wu (Queens University, Ontario, Canada) reported the use of
Quadrupole Central Transition (QCT) 17O NMR spectroscopy
to probe large protein-ligand complexes in solution.1 They
demonstrated that 17O-labeled ligands at ~30% 17O enrichment
can be detected in protein-ligand complexes of 240 kDa with
<1 mM protein concentrations, but expect the technique towork with complexes as large as 400-500 kDa when using 17O
enrichments as high as 90%. They also reported QCT 17O NMR
spectroscopy should be applicable to a wide variety of biologicalmacromolecules. Additionally, this laboratory reported the firstcomprehensive solid-state 17O MAS NMR of large protein-ligand
complexes.2

Catalog No.
OLM-782-90
OLM-782-85
OLM-782-70
OLM-782-60
OLM-782-40
OLM-782-20
OLM-782-10
NOLM-10743
NOLM-10742

Description
Water (17O, 90%)
Water (17O, 85%)
Water (17O, 70%)
Water (17O, 60%)
Water (17O, 35-40%)
Water (17O, 20%)
Water (17O, 10%)
L-Tyrosine (15N, 98%; carboxyl-17O2, 60%)
L-Valine (15N, 98%; carboxyl-17O2, 60%)

References
1.	Zhu, J.; Wu, G. 2011. Quadrupole central transition 17O NMR spectroscopy of
biological macromolecules in aqueous solution. J Am Chem Soc, 133(4), 920-932.
2.	Zhu, J.; Ye, E.; Terskikh, V.; Wu, G. 2010. Solid-state 17O NMR spectroscopyof large protein-ligand complexes. Angew Chem Int Ed, 49(45), 8399-8402.
3.	Metro, T.X.; Gervais, C.; Martinez, A.; et al. 2017. Unleashing the potential of17
O NMR spectroscopy using mechanochemistry. Angew Chem Int Ed, 56(24),6803-6807.

CIL is pleased to offer highly enriched 17O water as a starting
material to probe protein-ligand complexes using either QCT17
O NMR or solid-state 17O MAS NMR. Additionally, mechanochemistry has been shown to be a well-suited technique for17
O labeling of organic and inorganic compounds.3

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| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Miscellaneous Protein Reagents
Reverse Micelles

Reagents for Reverse Micelles

NMR spectroscopy of encapsulated proteins dissolved in
low viscosity fluids has emerged as a powerful supplement totraditional solution NMR approaches. Originally developed toovercome the slow tumbling problem presented by large soluble
proteins,1 the general approach of using reverse micelles has
now seen applications in the study of integral2 and anchored3
membrane proteins; proteins of marginal stability;4 protein
structure,5 dynamics 6 and hydration.7 Nucleic acids have also
been success­fully investigated in this manner.8 The distinguishingfeature of this approach is the nature of the sample. Spontaneously
formed reverse micelles are the dominant vehicle for encapsulationand the low-viscosity short-chain alkane fluids are the dominantsolvent with liquid propane and ethane being the most desirable.9
The availability of deuterated surfactants, cosurfactants and alkane
solvents avoids the complications of large unwanted 1H resonancesthat would greatly interfere with multidimensional NMR of
encapsulated biopolymers in low-viscosity solvents.

Catalog No.
DLM-276
DLM-8117
DLM-8840
DLM-691
DLM-1213
DLM-3476

Description
Ethane (D6, 98%)
Hexadecyltrimethylammonium bromide (D42, 98%)
Hexadecyltrimethylammonium chloride (D42, 98%)
n-Hexanol (D13, 98%)n-Pentane-D12 (D, 98%)
Propane (1,1,1,3,3,3-D6, 98%)

Deuterated Reagents
Catalog No.
DLM-710
DLM-54DLM-2
DLM-3
DLM-414
DLM-3908
DLM-2622
DLM-6686
DLM-2713

Description
Ammonium deuteroxide (D5, 99%) (~25% ND4OD in D2O)
Deuterium chloride (D, 99.96%) (~20% DCl in D2O)
Deuterium chloride (D, 99.5%) (~20% DCl in D2O)
Deuterium chloride (D, 99.5%) (~35% DCl in D2O)
EDTA (D12, 98%)
EDTA (D16, 98%)
DL-1,4-Dithiothreitol (DTT) (D10, 98%)
tris(2-Carboxyethyl)phosphine·DCl (TCEP) (D16, 98%)
2-Mercaptoethanol (D6, 98%)

References
1	
Wand, A.J.; Ehrhardt, M.R.; Flynn, P.F. 1998. High-resolution NMR of encapsulated
proteins dissolved in low-viscosity fluids. Proc Natl Acad Sci U S A, 95(26),15299-15302.
2	Kielec, J.M.; Valentine, K.G.; Babu, C.R.; et al. 2009. Reverse micelles
in integral membrane protein structural biology by solution NMR spectroscopy.
Structure, 17(3), 345-351.
3	Valentine, K.G.; Peterson, R.W.; Saad, J.S.; et al. 2010. Reverse micelle
encapsulation of membrane-anchored proteins for solution NMR studies.
Structure, 18(1), 9-16.
4	Peterson, R.W.; Anbalagan, K.; Tommos, C.; et al. 2004. Forced folding andstructural analysis of metastable proteins. J Am Chem Soc, 126(31), 9498-9499.
5	Babu, C.R.; Flynn, P.F.; Wand, A.J. 2001. Validation of protein structure frompreparations of encapsulated proteins dissolved in low-viscosity fluids.
J Am Chem Soc, 123(11), 2691-2692.

42

tel: +1.978.749.8000 | 1.800.322.1174 (USA) | fax: +1.978.749.2768

6	Simorellis, A.K.; Flynn, P.F. 2006. Fast local backbone dynamics of encapsulated
ubiquitin. J Am Chem Soc, 128(30), 9580-9581.
7	Nucci, N.V.; Pometun, M.S.; Wand, A.J. 2011. Site-resolved measurementof water-protein interactions by solution NMR. Nat Struct Mol Biol, 18(2), 245-249.
8	Workman, H.; Flynn, P.F. 2009. Stabilization of RNA oligomers through reverse
micelle encapsulation. J Am Chem Soc, 131(11), 3806-3807.
9	
Peterson, R.W.; Lefebvre, B.G.; Wand, A.J. 2005. High-resolution NMR studies of
encapsulated proteins in liquid ethane. J Am Chem Soc, 127(29), 10176-10177.

| Stable Isotopes for Biomolecular NMR

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Miscellaneous Protein ReagentsProtein Stabilization Reagent
Dr. Kaori Wakamatsu from Gunma University (Gunma, Japan)
has pioneered the use of choline-O-sulfate (COS) and uniformly
deuterated choline-O-sulfate (COS-D13) for use in protein NMR
studies. His results show significant improvement in stabilizing
soluble forms of protein in solution with a concurrent increase
in sensitivity in multidimensional NMR data sets acquired atnear-human physiological temperatures.

Advantages of COS:•	Prevention of thermal denaturation of membrane proteins,
including GPCRs•	Prevention of precipitation of protein and protein/peptide
complexes•	Facilitation of NMR measurements, especially at elevatedtemperatures• Improvement of protein recovery during purification
CIL is pleased to offer unlabeled and uniformly deuterated COS
to the worldwide protein NMR community.
Catalog No.
DLM-8937
ULM-8703

Description
Choline-O-sulfate (D13, 98%)
Choline-O-sulfate (unlabeled)Figure 1. 1D HSQC spectra of uniformly 15N-labeled Gi1α in the absence (left) orpresence of 1 M COS (right) were recorded on a Bruker ARX-400 spectrometer at 40°C
at 1 h (black), 17 h (red), 25 h (green), and 42 h (cyan) after temperature control. The
photographs demonstrate much less precipitate in the presence of COS at 42 h.

Figure 3. HSQC spectra of Gi1α labeled with [15N] phenylalanine in the presence of
1 M COS-d13 at 30°C (left) and 40°C (right). Almost all (18 out of 19) phenylalanine
signals are clearly observed at 40°C. Spectra were recorded on a Bruker Avance-700
spectrometer.

Figure 2. HSQC spectra of uniformly 15N labeled Gi1α in the presence of 1 M COS at
20°C (left) and 40°C (right). Spectra were recorded on a Bruker ARX-400 spectrometer.

Figure 4. COS prevents co-precipitation of Gi1α and its selective activator, m4I3C(14),on mixing. Gi1α and m4I3C(14) form precipitates on mixing (photograph at bottom,
left tube). The composition of the precipitates is confirmed by SDS-PAGE (bottom
right panel, lane ppt/0). In the presence of COS, the precipitates are not observed(photograph, SDS-PAGE).

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Cambridge Isotope Laboratories, Inc.

| Stable Isotopes for Biomolecular NMR

Protein NMR Standards
CIL offers labeled NMR standards that are used to test new pulse
sequences and monitor spectrometer performance.
Catalog No.
CNLM-2408

Description
GFL Peptide Standard (13C, 98%; 15N, 96-99%)
1 mM in DMSO-d6
Amino Acid Sequence
YGGFLRRI (bold indicates labeled residues)
CNLM-8663
His-GB1 (13C, 98%; 15N, 98%)
1.5 mM in PBS, pH 6.5, 0.02% sodium azide,
0.1 mM DSS, 10% D2O
Amino Acid Sequence
MHHHHHHGEN LYFQSMQYKL ILNGKTLKGE TTTEAVDAAT AEKVFKQYANDNGVDGEWTY DDATKTFTVT E

Don’t have the time or resources to express yourdesired protein? Ask your local CIL sales representative
about the possibility of supplying you with uniformly labeled,intact protein. Specify the following information: complete
protein sequence; type and location of a purification tag(if desired); labeling scheme; level of chemical purity; anddesired amount (in milligrams).

Also available in prepackaged 5 mM NMR tube; please inquire.

H,15N-HSQC of 1.5 mM immunoglobulin-binding fomain B1 of streptococcal protein G (13C, 99%; 15N, 99%) containing an N-terminal his6-tag and tobacco etch virus protease(TEV) cleavage site (catalog no. CNLM-8663-CA, Lot #20110209). The 15N-amide (top, right) and 1H-amide (bottom, right) assignments of CNLM-8663-CA show
excellent correlation with those previously reported in the Biological Magnetic Resonance Bank for GB1(BMRB #7280) lacking the his6-TEV leader sequence.
1

44

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| Stable Isotopes for Biomolecular NMR

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Protein Standards from Nexomics Biosciences, Inc.
CIL is pleased to offer Nexomics Biosciences, Inc. isotope-enriched
proteins for use as standards in NMR spectroscopy. Isotope-enriched
protein standards are ideal for:• Aiding in the development of new pulse sequences• Optimizing parameters for a given pulse sequence• Assessing spectrometer performance• Training purposesStandards are available with protein concentrations of 0.25 mM,
0.5 mM, and 1 mM. Please specify preferred concentration.
Standard packaging in an Eppendorf tube; 3 mM and 5 mM NMR
tubes are available upon request.

the biopharmaceutical community. Nexomics provides high-quality,
high-purity standards that are invaluable tools for bioNMR. Each
product is accompanied by the following data:• 1H-15N HSQC (15N-labeled proteins)• 1H-13C HSQC (13C-labeled proteins)•	CO-NH projection of 3D HNCO (15N,13C-labeled proteins)• SDS PAGE (for all labeled proteins)• MALDI-TOF (for all labeled proteins)• 15N-edited X-filtered 2D NOESY (NEX-XF1)

Nexomics is a New Jersey-based contract research organizationthat specializes in a broad array of gene-to-structure services to

Maltose-Binding Periplasmic Protein (MBP) from E. coli (strain K12) (residues 27-396)
NEX-MBP is a 40.84 kDa monomeric protein for which multiple sets of resonance assignments (BMRB database) and 3D structures(PDB database) are publicly available. As nonuniform sampling (NUS) and other NMR techniques emerge to push the size limitationsof NMR to new boundaries, large protein standards, such as NEX-MBP, will be required to test data-collection and processing strategies.

NEX-MBP1: Apo Conformation

NEX-MBP3: Open Conformation

Formulation: 20 mM sodium phosphate buffer (pH 7.2) in 10%
D2O and 0.02% NaN3
Number of residues: 370
MW (unlabeled): 40.84 kDa
Theoretical pI: 5.2
UniProt Accession: P0AEX9Formulation: 2 mM b-cyclodextrin, 20 mM sodium phosphatebuffer (pH 7.2) in 10% D2O and 0.02% NaN3
Number of residues: 370
MW (unlabeled): 40.84 kDa
Theoretical pI: 5.2
UniProt Accession: P0AEX9

Catalog No.
NEX-MBP1-U
NEX-MBP1-N
NEX-MBP1-CN-5
NEX-MBP1-CN
NEX-MBP1-CDN
NEX-MBP1-ILV
NEX-MBP1-ILVFY

Catalog No.
NEX-MBP3-U
NEX-MBP3-N
NEX-MBP3-CN-5
NEX-MBP3-CN
NEX-MBP3-CDN
NEX-MBP3-ILV
NEX-MBP3-ILVFY

Label
unlabeled(15N, 95%)
(13C, 5%; 15N, 95%)
(13C, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILV)(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILVFY)

Label
unlabeled(15N, 95%)
(13C, 5%; 15N, 95%)
(13C, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILV)(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILVFY)

NEX-MBP2: Closed Conformation
Formulation: 3 mM maltotriose, 20 mM sodium phosphate buffer(pH 7.2) in 10% D2O and 0.02% NaN3
Number of residues: 370
MW (unlabeled): 40.84 kDa
Theoretical pI: 5.2
UniProt Accession: P0AEX9
Catalog No.
NEX-MBP2-U
NEX-MBP2-N
NEX-MBP2-CN-5
NEX-MBP2-CN
NEX-MBP2-CDN
NEX-MBP2-ILV
NEX-MBP2-ILVFY

Label
unlabeled(15N, 95%)
(13C, 5%; 15N, 95%)
(13C, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILV)(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILVFY)

Protein Sequence
MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMEN
AQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITK

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| Stable Isotopes for Biomolecular NMR

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Protein Standards from Nexomics Biosciences, Inc. (continued)

13

C, 1H HSQC NEX-MBP3 “open” conformation.

CO-NH 2D plane of HNCO triple-resonance experiment of NEX-MBP2 “closed” sample.

46

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13

C, 1H HSQC “open” (blue) and “closed” (red) superposition.

SDS-PAGE GEL NEX-MBP (right).
NEX-MBP3 b-cyclodextrin complexed“open” sample (lane 1). NEX-MBP2
maltotriose complexed “closed”
sample (lane 2).

| Stable Isotopes for Biomolecular NMR

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X-Filtered NOESY NMR Standard (NEX-XF1)
In an X-filtered experiment, only NOEs between 15N/ 13C-1H and 14N/12C-1H (e.g., interchain NOEs) protons are observed. NOEs between
protons connected to 15N,13C are filtered (intrachain NOEs). When uniformly double-labeled protein sample is mixed with a naturalabundance protein sample, the interface will give rise to the only observable NOESY cross peaks. This powerful strategy enables the
spectroscopist to discern intra from inter NOESY cross peaks, thereby providing essential distance constraints for defining the dimer
interface (Lee, et al., 1994, 350, 87; Palmer, et al., 1991, 93, 151; Schleucher, et al., 1994, 4, 301).
NEX-XF1 is a 10.95 kDa protein (A. fulgidus antitoxin vapB21 homodimer) for which a set of resonance assignments (bmr7362),
3D structure (2NWT) and other NMR data are available in the public domain. This is a mixture of unlabeled and uniformly 15N/ 13Cenriched protein (25% homodimer unlabeled; 50% heterodimer unlabeled/labeled; 25% homodimer labeled) and is perfect to
set up X-filtered NOESY experiments.

NEX-XF1: Mixture of 25% homodimer unlabeled;
50% heterodimer unlabeled/labeled; 25% homodimer labeled
Formulation: 20 mM NH4OAc (pH 5.5), 100 mM NaCl, 5 mM
CaCl2, 10% D2O, 0.02 % NaN3
Number of residues: 38
MW (unlabeled): 10.95 kDa (homodimer)
Theoretical pI: 7.0
UniProt Accession: O28071

X-Filtered NOESY NMR Standard, His-Tagged
Catalog No.
NEX-XF1-HIS-CN

Label(13C, 95%; 15N, 95%)

2D 1H-1H plane of 1H, 13C edited 1H, 12C X-filtered NOESY.

Protein Sequence
MPKVIYENGVFKQKVDLKEGERVKIKLELKVE
PLEHHHHHH

SDS-PAGE GEL NEX-XF1 (right).

H-15N HSQC of NEX-XF1.

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| Stable Isotopes for Biomolecular NMR

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Protein Standards from Nexomics Biosciences, Inc. (continued)

Human Ubiquitin-60S Ribosomal Protein L40 (residues 1-76) (NEX-UB1)
NEX-UB1 is a small 8.8 kDa monomeric protein for which multiple sets of resonance assignments (BMRB database) and 3D structures(PDB database) are publicly available. Ubiquitin has been used as an industry-wide standard in the protein NMR field for many years.

NEX-UB1: After TEV cleavage
Formulation: 10 mM sodium phosphate buffer (pH 6.5) in
10% D2O, 0.02 % NaN3
Number of residues: 76
MW (unlabeled): 8.79 kDa
Theoretical pI: 6.7
UniProt Accession: P62987
Catalog No.
NEX-UB1-U
NEX-UB1-N
NEX-UB1-CN-5
NEX-UB1-CN
NEX-UB1-CDN
NEX-UB1-ILV
NEX-UB1-ILVFY

Label
unlabeled(15N, 95%)
(13C, 5%; 15N, 95%)
(13C, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILV)(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILVFY)

H, 15N HSQC of NEX-UB1.

1

NEX-UB1-HIS: With N-terminal His-Tag
Formulation: 10 mM sodium phosphate buffer (pH 6.5) in
10% D2O, 0.02 % NaN3
Number of residues: 90
MW (unlabeled): 10.59 kDa (with His-tag)
Theoretical pI: 6.7
UniProt Accession: P62987
Catalog No.
NEX-UB1-HIS-U
NEX-UB1-HIS-N
NEX-UB1-HIS-CN-5
NEX-UB1-HIS-CN
NEX-UB1-HIS-CDN
NEX-UB1-HIS-ILV
NEX-UB1-HIS-ILVFY

Label
unlabeled(15N, 95%)
(13C, 5%; 15N, 95%)
(13C, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILV)(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILVFY)

13

C-1H HSQC of NEX-UB1.

Protein Sequence after
TEV Cleavage
SHMQIFVKTLTGKTITLEVEPSDTIENVKAKIQD
KEGIPPDQQR LIFAGKQLEDGRTLSDY
NIQKESTLHLVLRLRGG

Protein Sequence before
TEV Cleavage
MGHHHHHHENLYFQSHMQIFVKTLTGKTITLEVEPSDTIEN VKAKIQDKEGIPPDQQRLIFAG
KQLEDGRTLSDYNIQKESTL HLVLRLRGG

SDS-PAGE GEL NEX-UB1 (right).

48

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CO-NH 2D plane of HNCO triple-resonance experiment of NEX-UB1.

| Stable Isotopes for Biomolecular NMR

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Immunoglobulin G-binding Protein G (GB1)
Formulation: 50 mM sodium phosphate (pH 5.5) in 10% D2O
and 0.02% NaN3
Number of residues: 56
MW (unlabeled): 6.22 kDa
Theoretical pI: 4.46
UniProt Accession: P06654
Catalog No.
NEX-GB1-U
NEX-GB1-N
NEX-GB1-CN-5
NEX-GB1-CN
NEX-GB1-CDN
NEX-GB1-ILV
NEX-GB1-ILVFY

Label
unlabeled(15N, 95%)
(13C, 5%; 15N, 95%)
(13C, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILV)(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILVFY)

Protein Sequence
MQYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTE

Calbindin-D9k (Protein S100-G)
Formulation: 50 mM ammonium acetate (pH 6.0) in 10% D2O
and 0.02% NaN3
Number of residues: 76
MW (unlabeled): 8.63 kDa
Theoretical pI: 4.53
UniProt Accession: P02633
Catalog No.
NEX-CAL-U
NEX-CAL-N
NEX-CAL-CN-5
NEX-CAL-CN
NEX-CAL-CDN
NEX-CAL-ILV
NEX-CAL-ILVFY

Label
unlabeled(15N, 95%)
(13C, 5%; 15N, 95%)
(13C, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILV)(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILVFY)

Protein Sequence
MKSPEELKGIFEKYAAKEGDPNQLSKEELKLLLQTEFPSLLKGPSTLDELFEELDKNGDGEVSFEEFQVLVKKISQ

Alpha-Spectrin SH3 Domain
Formulation: 10 mM sodium citrate (pH 3.5) in 10% D2O
and 0.02% NaN3
Number of residues: 71
MW (unlabeled): 8.39 kDa
Theoretical pI: 6.64
UniProt Accession: P07751
Catalog No.
NEX-SH3-U
NEX-SH3-N
NEX-SH3-CN-5
NEX-SH3-CN
NEX-SH3-CDN
NEX-SH3-ILV
NEX-SH3-ILVFY

Label
unlabeled(15N, 95%)
(13C, 5%; 15N, 95%)
(13C, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15N, 95%)
(13C, 95%; D, 95%; 15 N, 95%; 13CH3-ILV)(13C, 95%; D, 95%; 15N, 95%; 13CH3-ILVFY)

Protein Sequence
MGHHHHHHSHMDETGKELVLALYDYQEKSPREVTMKKGDILTLLNSTNKDWWKVEVNDRQF
VPAAYVKKLD

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| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Nucleic Acids
Although solution-state NMR is a powerful and versatile tool
for studying the structure and dynamics of RNA and DNA, using
13
C correlation spectroscopy to determine structure of RNA in thesolid-state1 shows promise. The different types of information that
may be gained from NMR studies of RNA and DNA2 include basepairing patterns, conformational equilibria, site-specific information
regarding ligand binding, delineation of secondary structure motifs
such as hairpins and bulges, local structure and dynamics, andglobal structure derived from residual dipolar couplings.
The most popular approaches to produce labeled RNA molecules
for NMR studies use enzymatic in vitro transcription methods that
employ labeled rNTPs, T7 RNA polymerase, and either linearized
plasmids or double-stranded DNA as templates. These techniques
are used to construct labeled RNA molecules of which all of one
type of nucleotide is labeled. Perdeuterated NTPs can be used in
combination with protonated NTPs to create RNA molecules inwhich specific types of nucleotides are protonated, thus allowing
spectral editing without the significant signal broadening associatedwith 13C incorporation.3
Because severe signal degeneracy has hampered NMR studies oflarger RNAs, key researchers in this area have utilized selectively
deuterated rNTPs, in conjunction with in vitro synthesis methods,
to reduce spectral complexity, spectral line-widths, and forobserving NOEs over larger distances.3
Labeled DNA oligonucleotides are routinely synthesized using
enzymatic in vitro methods that utilize labeled dNTPs, a DNA
polymerase, and a cDNA template. One advantage of using
enzymatic methods over phosphoramidite chemistry is that large
oligonucleotides (e.g., >50 nucleotides in length) can be preparedin milligram quantities. Position-specific labeled DNA molecules canbe synthesized using standard phosphoramidite chemistry (using
CIL’s deoxyphosphoramidites) to overcome the limited chemicalshift dispersion of DNA, as well as to create residue-specific probes
to obtain functional, structural and dynamic information.

Cassia LLC was founded in 2005 by noted NMR spectroscopists
Drs. Jamie Williamson and Lincoln Scott. CIL and Cassia havea special relationship that combines CIL’s isotopic material
production and marketing with Cassia’s unique expertise in
RNA and DNA biosynthesis. Since 2005, CIL and Cassia havedeveloped the most extensive product line of stable isotopelabeled RNA and DNA triphosphates, DNA phosphoramidites
and other related compounds. All of these products areroutinely available for immediate shipment from stockat CIL.

Top 10 Reasons to Use Ammonium Salts
1.

Self-buffering (pH ~7.6).

2.

“Soft cation.”

3.	Nucleotides of ammonium salts are active withpolymerases, synthetases, and phosphatases.
4.

Volatile counter-ion.

5.	The ammonium cation can be easily exchanged
using DOWEX cation exchange resin.
6.	The pH does not change during drying of thenucleotide (i.e. “speed-vac,” lyophilize).
7.	Stoichiometry between the counter-ion and thenucleotide is preserved.
8.

Routinely compatible in down-stream syntheses.

9.	Compatible in a variety of down-stream
chromatography applications.
10.	Tested to be comparable in side-by-side in vitrotranscription reactions!

CIL is pleased to offer labeled mono- and triphosphateribonucleotides, deoxyribonucleotides, and phosphoramidites
for use in NMR investigations.

References
1.	Cherepanov, A.V.; Glaubitz, C.; Schwalbe, H. 2010. High-resolution studies of
uniformly 13C,15N-labeled RNA by solid-state NMR spectroscopy. Angew ChemInt Ed Engl, 49(28), 4747-4750.
2.	Furtig, B.; Wohnert, C.; Schwalbe, H. 2003. NMR spectroscopy of RNA.
Chembiochem, 4(10), 936-962.
3.	Lu, K.; Miyazaki, Y.; Summers, M. 2010. Isotope labeling strategies for NMR
studies of RNA. J Biomol NMR, 46(1), 113-125.

❛❛We’ve been using CIL’s perdeuterated rNTPs for many years
to facilitate NMR studies of larger RNAs. Their newer
products, which include partially deuterated rNTPs, havebeen extraordinarily helpful, and have enabled highresolution NMR structural studies of RNAs that werepreviously intractable.❜❜Michael Summers, PhD
Howard Hughes Medical Institute
University of Maryland, Baltimore County

50

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| Stable Isotopes for Biomolecular NMR

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Ribonucleic Acids
Ribonucleoside Triphosphates (13C, 15N, 13C/15N, D)
Catalog No.
NEW! CLM-8426-CA
NEW! CLM-10987-CA
NEW! CLM-10988-CA
NEW! CLM-10914-CA

NLM-3987-CA
NLM-4266-CA
NLM-4268-CA
NLM-4270-CA
NLM-7519-CA
CNLM-4265-CA
CNLM-4267-CA
CNLM-4269-CA
CNLM-4271-CA
CNLM-7503-CA
DLM-7514-CA
DLM-7515-CA
DLM-7516-CA
DLM-7517-CA
DLM-7518-CA

Description
Adenosine 5’-triphosphate, ammonium salt (13C10, 99%) (in solution) CP 95%
Cytidine 5’-triphosphate, ammonium salt (13C9, 99%) (in solution) CP 95%
Guanosine 5’-triphosphate, ammonium salt (13C10, 99%) (in solution) CP 90%
Uridine 5’-triphosphate, ammonium salt (13C9, 99%) (in solution) CP 90%
Adenosine 5’-triphosphate, ammonium salt (15N5; 98-99%) (in solution) CP 90%
Cytidine 5’-triphosphate, ammonium salt (15N3; 96%) (in solution) CP 90%
Guanosine 5’-triphosphate, ammonium salt (15N5; 98-99%) (in solution) CP 90%
Uridine 5’-triphosphate, ammonium salt (15N2; 98-99%) (in solution) CP 90%
Set of 4 Ribonucleoside 5’-triphosphates (15N, 98%) (ammonium salt/ in solution) CP 90%
Adenosine 5’-triphosphate, ammonium salt (13C, 15N; 98-99%) (in solution) CP 90%
Cytidine 5’-triphosphate, ammonium salt (13C, 15N; 96-98%) (in solution) CP 90%
Guanosine 5’-triphosphate, ammonium salt (13C, 15N; 98-99%) (in solution) CP 90%
Uridine 5’-triphosphate, ammonium salt (13C, 15N; 98-99%) (in solution) CP 90%
Set of 4 Ribonucleoside 5’-triphosphates (13C, 15N; 98-99%) (ammonium salt/ in solution) CP 90%
Adenosine 5’-triphosphate, ammonium salt (D8, 97%) (in solution) CP 90%
Cytidine 5’-triphosphate, ammonium salt (D8, 97%) (in solution) CP 90%
Guanosine 5’-triphosphate, ammonium salt (D7, 97%) (in solution) CP 90%
Uridine 5’-triphosphate, ammonium salt (D8, 97%) (in solution) CP 90%
Set of 4 Ribonucleoside 5’-triphosphates (D, 98%) (ammonium salt/ in solution) CP 90%

Please inquire for lithium salts.
References
Song, Y.; Marmion, R.A.; Park, J.O.; et al. 2017. Dynamic control of dNTP synthesis in early embryos. Dev Cell, 42(3), 301-308.
Le, M.T.; Brown, R.E.; Simon, A.E.; et al. 2015. In vivo, large-scale preparation of uniformly N-15 and site-specifically C-13 labeled homogeneous, recombinant RNA for NMR studies.
Methods Enzymol, 565, 495-535.
Alverado, L.J.; Longhini, A.P.; LeBlanc, R.M.; et al. 2014. Chemo-enzymatic synthesis of selectively 13C/ 15N labeled RNA for NMR structural and dynamics studies. Methods Enzymol,
549, 133-162.
Lu, K.; Miyazaki, Y.; Summers, M.F. 2010. Isotope labeling strategies for NMR studies of RNBA. J Biomol NMR, 46(1), 113-125.
Dayie, K.T. 2008. Key labeling technologies to tackle sizeable problems in RNA structural biology. Int J Mol Sci, 9(7), 1214-1240.
Nikonowicz, E.P.; Sirr, A.; Legault, P.; et al. 1992. Preparation of C-13 and N-15 labeled RNAs for heteronuclear multidimensional NMR studies. Nucleic Acids Res, 20(17), 4507-4513.

Fluorine-Labeled Nucleotide 5’-Triphosphates
The fluorine nucleus is extremely sensitive to the local chemical
environment, which leads to a wide chemical shift range. This
makes it an excellent probe for secondary structure, especiallywhere the chemical shift dispersion is limited, such as in RNA.
Although researchers have used 19F-NMR to study nucleic acids
for decades, only recently has a larger RNA been uniformlylabeled with fluorine and investigated using 19F-NMR.1
CIL is pleased to offer new fluorinated nucleotide 5’-triphosphates
for use in studying structure and dynamics of RNA using 19F-NMR.

New!

Catalog No.
ULM-10696-CA
ULM-10697-CA
ULM-10698-CA

Description
5-Fluorouridine 5’-triphosphate (5F-UTP)
5-Fluorocytidine 5’-triphosphate (5F-CTP)
2-Fluoroadenosine 5’-triphosphate (2F-ATP)❛❛When my lab makes labeled RNA, we count on high yields
and purity. We have been using 13C/15N ribonucleotide
triphosphates (rNTPs) and selectively deuterated rNTPs from

References

CIL for the past 15 years. We have always been very happy

1.	Sochor, F.; Silvers, R.; Muller, D.; et al. 2016. 19F-labeling of the adenine H2-site
to study large RNAs by NMR spectroscopy. J Biomol NMR, 64(1), 63-74.

with the performance and quality of the CIL rNTPs. They are
the gold standard.❜❜Samuel Butcher, PhD
Department of Biochemistry
University of Wisconsin-Madison

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

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| Stable Isotopes for Biomolecular NMR

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Ribonucleic Acids
Ribonucleoside Triphosphates (Selectively Labeled D)
Catalog No.
DLM-7862
DLM-8815-CA
DLM-9619-CA
DLM-9366-CA
DLM-9267-CA
DLM-8594-CA
DLM-9365-CA
DLM-9100-CA
DLM-8637-CA

Description
Equimolar Mix: ATP, GTP, (ribose-3’,4’,5’,5”-D4, 98%); CTP, UTP (5-D, ribose-3’,4’,5’,5”-D4, 98%) NH4+ salt (see structures below)
Adenosine 5’-triphosphate, ammonium salt (2-D, 97%) (in solution) CP 90%
Adenosine 5’-triphosphate, ammonium salt (ribose-1’,2’,3’,4’,5’,5’’-D6, 98%) (in solution) CP 90%
Cytidine 5’-triphosphate, ammonium salt (cytosine-5-D, 97%) (in solution) CP 90%
Cytidine 5’-triphosphate, ammonium salt (5,6-D2, 97%) (in solution) CP 90%
Cytidine 5’-triphosphate, ammonium salt (cytosine-5-D, 6-H; ribose-1,2,3,4,5,5-D6, 96-97%) (in solution)
Uridine 5’-triphosphate, ammonium salt (uracil-5-D, 98%) (in solution) CP 90%
Uridine 5’-triphosphate, ammonium salt (5,6-D2, 98%) (in solution) CP 90%
Uridine 5’-triphosphate, ammonium salt (uracil-5-D, 6-H; ribose-1,2,3,4,5,5-D6, 96-97%) (in solution)

DLM-8922

DLM-8922-CA
DLM-8924-CA
DLM-8923-CA
DLM-8925-CA

DLM-8924

DLM-8923

DLM-8925

Adenosine-5’-triphosphate, ammonium salt (ribose-3’,4’,5’,5’’-D4, 98%) (in solution)
Cytidine-5’-triphosphate, ammonium salt (5-D, ribose-3’,4’,5’,5’’-D4, 98%) (in solution)
Guanosine-5’-triphosphate, ammonium salt (ribose-3’,4’,5’,5’’-D4, 98%) (in solution)
Uridine-5’-triphosphate, ammonium salt (5-D, ribose-3’,4’,5’,5’’-D4, 98%) (in solution)

H-1H-NOESY Spectra of the Tetraloop-Receptor RNA (45 nt dimer; 30 kDa). Spectra above are from unlabeled RNA (a, c, e) and selectively deuterated RNA (b, d, f).
The selectively deuterated RNA was prepared using the equimolar mix (CIL catalog no. DLM-7862). The left two panels (a, b) contain NOEs between the H1’ proton and all otherribose protons. The middle two panels (c, d) contain NOEs between the base protons and H1’ protons. The right two panels (e, f) contain NOEs between the base and other ribose
protons. Spectra taken from Davis, J.H., et al. (see reference below) were provided courtesy of professor Sam Butcher at the University of Wisconsin. The sequential assignment
pattern of inter- and intranucleotide NOEs is shown for the D5-RNA. The advantages of the selectively deuterated pattern are evident in these key regions of the spectra.
1

Reference
Davis, J.H.; Tonelli, M.; Scott, L.G.; et al. 2005. Helical packing in solution: NMR
structure of a 30 kDa GAAA tetraloop-receptor complex. J Mol Biol, 351(2), 371-382.

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

52

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| Stable Isotopes for Biomolecular NMR

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Ribonucleic Acids
Ribonucleoside Monophosphates (15N, 13C/15N)
Catalog No.
NLM-3792
NLM-3793
NLM-3794
NLM-3795
CNLM-3802
CNLM-3803
CNLM-3804
CNLM-3805

Description
Adenosine 5’-monophosphate, lithium salt (15N5, 96-98%) (in solution)
Cytidine 5’-monophosphate, lithium salt (15N3, 96-98%) (in solution)
Guanosine 5’-monophosphate, lithium salt (15N5, 96-98%) (in solution)
Uridine 5’-monophosphate, lithium salt (15N2, 96-98%) (in solution)
Adenosine 5’-monophosphate, lithium salt (13C10, 98%; 15N5, 96-98%) (in solution)
Cytidine 5’-monophosphate, lithium salt (13C9, 98%; 15N3, 96-98%) (in solution)
Guanosine 5’-monophosphate, lithium salt (13C10, 98%; 15N5, 96-98%) (in solution)
Uridine 5’-monophosphate, lithium salt (13C9, 98%; 15N2, 96-98%) (in solution) CP 90%

Deoxyribonucleic Acids
Deoxyribonucleoside Triphosphates (15N, 13C/15N, D)
NLM-6217-CA

2’-Deoxyguanosine 5’-triphosphate, ammonium salt (15N5; 98-99%) (in solution) CP 90%

NLM-6215
NLM-6216
NLM-6217
NLM-6218
NLM-75122’-Deoxyadenosine 5’-triphosphate, lithium salt (15N5, 98%) (in solution) CP 90%
2’-Deoxycytidine 5’-triphosphate, lithium salt (15N3, 98%) (in solution) CP 90%
2’-Deoxyguanosine 5’-triphosphate, lithium salt (15N5, 98%) (in solution) CP 90%
Thymidine 5’-triphosphate, lithium salt (15N2, 98%) (in solution) CP 90%
Set of 4 2’-Deoxyribonucleoside 5’-triphosphates (15N, 98%) (lithium salt /in solution) CP 90%

CNLM-6219-CA
CNLM-6221-CA

2’-Deoxyadenosine 5’-triphosphate, ammonium salt (13C10, 98%; 15N5, 97-98%) CP 90%
2’-Deoxyguanosine 5’-triphosphate, ammonium salt (13C10, 98%; 15N5, 96-98%) CP 90%

CNLM-6219
CNLM-6220
CNLM-6221
CNLM-6222
CNLM-7513

2’-Deoxyadenosine 5’-triphosphate, lithium salt (13C10, 98%; 15N5, 98%) (in solution) CP 90%
2’-Deoxycytidine 5’-triphosphate, lithium salt (13C9, 98%; 15N3, 98%) (in solution) CP 90%
2’-Deoxyguanosine 5’-triphosphate, lithium salt (13C10, 98%; 15N5, 98%) (in solution) CP 90%
Thymidine 5’-triphosphate, lithium salt (13C10, 98%; 15N2, 98%) (in solution) CP 90%
Set of 4 2’-Deoxyribonucleoside 5’-triphosphates (13C, 98%; 15N, 98%) (lithium salt /in solution) CP 90%

DLM-7507
DLM-7508
DLM-7509
DLM-7510
DLM-7511

2-Deoxyadenosine 5’-triphosphate, lithium salt (D, 97%) (in solution) CP 90%
2-Deoxycytidine 5’-triphosphate, lithium salt (D, 97%) (in solution) CP 90%
2-Deoxyguanosine 5’-triphosphate, lithium salt (D, 97%) (in solution) CP 90%
Thymidine 5’-triphosphate, lithium salt (D, 97%) (in solution) CP 90%
Set of 4 2-Deoxyribonucleoside 5’-triphosphates (D, 98%) (lithium salt /in solution) CP 90%

Deoxyribonucleoside Monophosphates (15N, 13C/15N)
NLM-3919
NLM-3921
NLM-6835
NLM-3925
CNLM-3918
CNLM-6834
CNLM-6836
CNLM-3924
CNLM-7871

2’-Deoxyadenosine 5’-monophosphate, lithium salt (15N5, 98%) (in solution)
2’-Deoxycytidine 5’-monophosphate, lithium salt (15N3, 96%) (in solution)
2’-Deoxyguanosine 5’-monophosphate, lithium salt (15N, 98%) (in solution)
Thymidine 5’-monophosphate, lithium salt (15N2, 98%) (in solution)
2’-Deoxyadenosine 5’-monophosphate, lithium salt (13C10, 98%; 15N5, 98%) (in solution)
2’-Deoxycytidine 5’-monophosphate, lithium salt (13C, 98%; 15N, 98%) (in solution)
2’-Deoxyguanosine 5’-monophosphate, lithium salt (13C, 98%; 15N, 98%) (in solution)
Thymidine 5’-monophosphate, lithium salt (13C10, 98%; 15N2, 98%) (in solution)
Set of 4 2’-Deoxyribonucleoside 5’-monophosphates (13C, 98%; 15N, 98%) (lithium salt /in solution)❛❛W ithout these labeled rNTPs from CIL we would not havebeen able to prepare such high-quality samples which madethe assignment and structure determination possible.❜❜Michael Durney, PhD
Department of Molecular and Cellular Biology
Harvard University

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

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| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Deoxyribonucleic Acids
Deoxyribophosphoramidites (13C/15N)
Catalog No.
CNLM-6828
CNLM-6830
CNLM-6825
CNLM-6824

Description
2’-Deoxyadenosine phosphoramidite (13C10, 98%; 15N5, 98%) CP 95%
2’-Deoxycytidine phosphoramidite (13C9, 98%; 15N3, 98%) CP 95%
2’-Deoxyguanosine phosphoramidite (13C10, 98%; 15N5, 98%) CP 95%
Thymidine phosphoramidite (13C10, 98%; 15N2, 98%) CP 95%
O

O

*
HN

OCH3

*
H3CO

O

*

*

O

*

O
H3C

*
N
* *

*
N

*N * N
*

*

*

P

OCH3

H3CO

*

*

O

*

O

CN

CH3 CH3

*

P

H3CO

O

*

*

*

CH3

CN

P

O

*
*N * N * N
H
*

H3CO

CH3

*

CN

*
N

O

*

*

O

P

H3C

CH3 CH3

CNLM-6830

*

O

O

*

O

*
CH
3

* * *
HN

CH3
CH3

*

N

O

OCH3

N *
*
* NH

*

*

H 3C

CH3 CH3

CNLM-6828

O

O

O

N

H 3C

*
O

O

*

*
N

* N*
*

CH3

OCH3

*

*

O

*

O

N

*

HN

*

*

O

CH3

N

CN

CH3 CH3

CNLM-6825

CNLM-6824

Deoxyribophosphoramidites (15N)
NLM-6829
NLM-6827
NLM-6826
NLM-6823

2’-Deoxyadenosine phosphoramidite (15N5, 98%) CP 95%
2’-Deoxycytidine phosphoramidite (15N3, 98%) CP 95%
2’-Deoxyguanosine phosphoramidite (15N5, 98%) CP 95%
Thymidine phosphoramidite (15N2, 96-98%) CP 95%
O

O
OCH3

H3CO

*
N
*N

O

O
O

H3C

*
HN

P

O

N

CH3

OCH3

*
N
N

*

*

H3CO

*

*

N

P

CH3

CN

CH3 CH3

NLM-6827❛❛CIL has been a strong supporter of NMR methods ofdevelopment over the years, providing critical isotopeenriched reagents for research and development, without
which many of the recent advances in biomolecular NMR
would simply not have been possible. In particular, the broad
biological impact and tremendous success of the multidimensional triple-resonance biomolecular NMR would nothave been achieved without the high quality and broadly
accessible reagents that CIL has provided to the scientificcommunity over the last 35 years.❜❜
Gaetano Montelione, PhD
Distinguished Professor of Molecular Biology
and Biochemisty, Rutgers University
Founder, Nexomics Biosciences

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

54

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H3C

*
NH

*N

O

N

*

O
O

O

N

H3C

NLM-6829

H3CO

O
O

CH3 CH3

O

O

*

N

N

O

CN

OCH3

HN

P

N

O
CH3

CH3 CH3

NLM-6826

*
N
H

O

OCH3
O

CH3
CH3

H3CO

O

*
N

O
O
O

CN

CH3

*
HN

H3C

P

N

O
CH3

CH3 CH3

NLM-6823

CN

| Stable Isotopes for Biomolecular NMR

isotope.com

Miscellaneous RNA and DNA Products
Catalog No.
CLM-1654
NLM-6924
CLM-3698
CLM-7674
CLM-3678
DLM-7676
DLM-7677
DLM-7678
NLM-9750
CNLM-3806-CA
CLM-3605
CLM-3611
CLM-3699
CLM-3679
DLM-7681
NLM-3797
CNLM-3807
CNLM-4424
NLM-3895
CLM-3700
CLM-3701
CLM-7682
CLM-4579
DLM-7683
CNLM-3896-CA
NLM-3897
CNLM-3898
CLM-7684
CLM-3702
DLM-7685
DLM-6142
CLM-7686
DLM-7687
NLM-3899-CA
CNLM-3900-CA
NLM-6715
CNLM-8771-CA
CNLM-10422
DLM-4391
CNLM-4510

Description
Adenine (8-13C, 95%)
Adenine·HCl (15N5, 98%)
Adenosine (ribose-2-13C, 99%)
Adenosine (3’-13C, 98%)
Adenosine (ribose-13C5, 98%) CP 97%
Adenosine (ribose-1-D, 98%)
Adenosine (ribose-2-D, 97%)
Adenosine (ribose-5,5-D2, 98%)
Adenosine (15N5, 96-98%)
Adenosine (13C10, 98%; 15N5, 96-98%)
Adenosine·H2O (ribose-1-13C, 99%) CP 95%
Cytidine (ribose-1-13C, 99%)
Cytidine (ribose-2-13C, 99%)
Cytidine (ribose-13C5, 98%)
Cytidine (ribose-5,5-D2, 98%)
Cytidine (15N3, 96-98%)
Cytidine (13C9, 98%; 15N3, 96-98%)
Cytosine (2-13C, 99%; 1,3-15N2, 98%)
2’-Deoxyadenosine (15N5, 96-98%)
2’-Deoxyadenosine·H2O (deoxyribose-1-13C, 99%)
2’-Deoxyadenosine·H2O (deoxyribose-2-13C, 99%)
2’-Deoxyadenosine·H2O (ribose-5-13C, 98%)
2’-Deoxyadenosine·H2O (ribose-13C5, 99%)
2’-Deoxyadenosine·H2O (ribose-5,5-D2, 98%)
2’-Deoxyadenosine·H2O (13C10, 98%; 15N5, 96-98%)
2’-Deoxycytidine (15N3, 96-98%)
2’-Deoxycytidine (13C9, 98%; 15N3, 96-98%)
2’-Deoxycytidine·H2O (ribose-1-13C, 98%)
2’-Deoxycytidine·H2O (deoxyribose-2-13C, 99%)
2’-Deoxycytidine·H2O (ribose-5,5-D2, 98%)
5-Methyl-2’-deoxycytidine·HCl(methyl-D3, ring-6-D, 96-98%) CP 95%
2’-Deoxyguanosine·H2O (ribose-1-13C, 98%)
2’-Deoxyguanosine·H2O (ribose-5,5-D2, 98%)
2’-Deoxyguanosine·H2O (15N5, 98%) CP 95%
2’-Deoxyguanosine·H2O (13C10, 98%; 15N5, 96-98%)
8-Hydroxy-2’-deoxyguanosine (15N5, 98%) CP 95%
2’-Deoxyuridine,·ammonium salt
(13C9, 98-99%; 15N2, 98-99%) (in solution) CP 90%
2’-Fluoro-2’-deoxyuridine (uracil-13C4, 99%; 15N2, 98%)
5,6-Dihydrothymine (5,6,6-D3, methyl-D3, 95%)
5,6-Dihydrouracil (13C4, 99%; 15N2, 98%)

Catalog No.

Description

NLM-798
CNLM-3916
CLM-1019
NLM-6925
CNLM-3990
NLM-3798
CLM-7688
CNLM-3808-CA
DLM-7689
CNLM-3832
CNLM-4392
DLM-7471
DLM-7473
DLM-7472
CNLM-10505
CLM-3629
NLM-10691
CLM-3647
CLM-4289
CLM-3703
CLM-7692
DLM-7691
NLM-3901
CNLM-3902
DLM-1089
NLM-3995
CNLM-6945
CLM-3276
CLM-692
CLM-10507
DLM-8633
DLM-8502
NLM-637
CNLM-3917
CLM-3630
CLM-3680
DLM-7693
NLM-812
CNLM-3809

5-Fluorouracil (1,3-15N2, 99%)5-Fluorouracil (13C4, 99%; 15N2, 98%)
Guanine (8-13C, 98%)
Guanine (15N5, 98%)
Guanine (8-13C, 98%; 7,9-15N2, 98%)
Guanosine (15N5, 96-98%)
Guanosine·H2O (ribose-1-13C, 98%)
Guanosine·H2O (13C10, 98%; 15N5, 96-98%)
Guanosine·H2O (ribose-5,5-D2, 98%)
8-Hydroxyadenine (8-13C, 98%; 6,9-diamino-15N2, 98%)
5-Hydroxycytosine (2-13C, 99%; 1,3-15N2, 98%)
3-Methyladenine (methyl-D3, 98%)
6-O-Methylguanine (methyl-D3, 98%)
7-Methylguanine (methyl-D3, 98%)
2-Pyrimidinone (2-13C, 99%; 15N2, 98%)
Ribothymidine (ribose-1-13C, 99%)
a-Thymidine (15N2, 98%)
Thymidine (methyl-13C, 98%)
Thymidine (deoxyribose-1-13C, 99%)
Thymidine (deoxyribose-2-13C, 99%)
Thymidine (deoxyribose-3-13C, 99%)
Thymidine (deoxyribose-5,5-D2, 98%)
Thymidine (15N2, 96-98%) CP 97%
Thymidine (13C10, 98%; 15N2, 96-98%)
Thymine (a,a,a,6-D4, 98%)
Thymine (1,3-15N2, 98%)
Thymine (13C5, 98%; 15N2, 98%)
Uracil (2-13C, 99%)
Uracil (4,5-13C2, 99%)
Uracil (13C4, 99%)
Uracil (5-D, 98%)
Uracil (5,6-D2, 98%)
Uracil (1,3-15N2, 98%)
Uracil (13C4, 99%; 15N2, 98%)
Uridine (ribose-1-13C, 99%)
Uridine (ribose-13C5, 98%)
Uridine (ribose-5,5-D2, 98%)
Uridine (15N2, 98%)
Uridine (13C9, 98%; 15N2, 96-98%)

Chemical purity (CP) is 98% or greater, unless otherwise specified.
For research use only. Not for use in diagnostic procedures.

cilsales@isotope.com | intlsales@isotope.com | isotope.com 55

| Stable Isotopes for Biomolecular NMR

Cambridge Isotope Laboratories, Inc.

Deuterated Buffers
Catalog No.
DLM-12

Description
Acetic acid-D4 (D, 99.5%)

DLM-41
DLM-1099
DLM-1273
DLM-710
DLM-407
DLM-4528
DLM-4862
DLM-286
DLM-280

Acetic acid-D4 “100%” (D, 99.91%)
Ammonium bromide (D4, 98%)
Ammonium chloride (D4, 98%)
Ammonium deuteroxide (D5, 99%) ~25% in solution D2O
Betaine (D11, 98%)
Bis-tris (D19, 98%)
Cacodylic acid (D7, 98%)
Formic acid (D2, 98%) <5% D2OGlycine (D5, 98%)

Catalog No.
DLM-3786
DLM-3033
DLM-4363
DLM-4781
DLM-1361
DLM-831
DLM-1842
DLM-4779
DLM-1814
DLM-3593

Description
HEPES (D18, 98%)
Imidazole (D4, 98%)
MES (D13, 98%)
PIPES (D18, 98%)
Sodium formate (D, 98%)
Succinic acid (D6, 98%)
Tricine (D8, 98%)
Trimethylamine N-oxide (D9, 98%)
TRIS (D11, 98%)
TRIS (D11, 98%) 1 M in D2O

pH Buffering Range Chart
pKa is indicated by a white rule within the range

Compound and Catalog Number
Acetic acid-d4 (D, 99.5%) DLM-12

4.3 –5.2

Acetic acid-d4 “100%” (D, 99.91%) DLM-41

4.3 –5.2

Ammonium bromide (D4, 98%) DLM-1099

8.8 –9.7

Ammonium chloride (D4, 98%) DLM-1273

8.8 –9.7

Ammonium deuteroxide (D5, 99%) DLM-710

8.8 –9.7

Betaine (D11, 98%) (trimethylglycine) DLM-407

1.4 –2.3

Bis-tris (D19, 98%) DLM-4528

5.8 –7.2

Cacodylic acid (D7, 98%) (dimethylarsinic acid) DLM-4862

5.1 –7.1

Formic acid (D2, 98%) (contains < 5% D2O) DLM-286

3.3 –4.2

Glycine (D5, 98%) DLM-280

9.1 –10.1

1.9 –2.8

N-(2-Hydroxyethyl)piperazine-N’-2-ethanesulfonicacid (D18, 98%) (HEPES) DLM-3786

6.8 –8.2

Imidazole (D4, 98%) DLM-3033

6.6 –7.5

2-(N-morpholino)ethanesulfonic acid (D13, 98%) (MES)
DLM-4363

5.5 –6.7

Piperazine-N,N’-bis(2-ethanesulfonic acid)(D18, 98%) (PIPES) DLM-4781

6.1 –7.5

Sodium formate (D, 99%) DLM-1361

3.3 –4.2

Succinic acid (D6, 98%) DLM-831

5.0 –5.9

3.8–4.6

Tricine (D8,98%) DLM-1842

7.6 –8.8

Tris(hydroxymethyl)methylamine (D11, 98%)
(TRIS) DLM-1814

7.8–8.8

Tris(hydroxymethyl)methylamine (D11, 98%)
(TRIS) (1 M solution in D2O) DLM-3593

7.8–8.8

1

1.5 2

2.5 3

3.5 4

4.5 5

5.5 6

6.5 7

pH Buffering Range

56

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7.5 8

8.5 9

9.5 1 0 10.51 1

| Stable Isotopes for Biomolecular NMR

isotope.com

Solvent Data Chart
H Chemical shift(ppm from TMS)
(multiplicity)�1Acetone-d 6

11.65 (1)2.04 (5)2.05 (5)2.2
2.2

Acetonitrile-d3

1.94 (5)2.5

Acetic acid-d4

Benzene-d6
Chloroform-d
Cyclohexane-d12
Deuterium oxide
N,N-Dimethyl-formamide-d7

Dimethyl sulfoxide-d6
1,4-Dioxane-d 8
Ethanol-d6

Methanol-d4
Methylene chloride-d2
Pyridine-d5

1,1,2,2-Tetrachloroethane-d2
Tetrahydrofuran-d8

7.16 (1)
7.24 (1)1.38 (1)4.80 (DSS)
4.81 (TSP)
8.03 (1)2.92 (5)2.75 (5)2.50 (5)3.53 (m)5.19 (1)3.56 (1)1.11 (m)4.87 (1)
3.31 (5)5.32 (3)8.74 (1)
7.58 (1)
7.22 (1)
6.0
3.58 (1)1.73 (1)

1.9
1.9
1.9

1.7
1.1

Toluene-d8

Trifluoroacetic Acid-d
Trifluoroethanol-d3

7.09 (m)7.00 (1)
6.98 (5)2.09 (5)11.50 (1)5.02 (1)3.88 (4x3)

C Chemical shift(ppm from TMS)
(multiplicity)
�

JCD
(Hz)

H Chemical shiftof HOD(ppm from TMS)�

Density at
20ºC
�Melting point(ºC)
�Boiling point(ºC)
�Dielectric
constant(unlabeled)

Molecular
weight�

178.99 (1)20.0 (7)206.68 (1)29.92 (7)118.69 (1)1.39 (7)128.39 (3)77.23 (3)26.43 (5)
NA

20.0

11.5*

1.12

16.7

118

6.1

64.08

00.9
19.4

2.8*

0.87

-94

56.5

20.7

64.12

2.1*

0.84

-45

81.6

37.5

44.07

0.4*
1.5*
0.8*
4.8*

0.95
1.50
0.89
1.11

5.5
-63.5
6.47
3.81

80.1
61-62
80.7
101.42

2.3
4.8
2.0
78.5

84.15
120.3896.24
20.03

163.15 (3)34.89 (7)29.76 (7)39.51 (7)66.66 (5)

29.4
21.0
21.1
21.0
21.9

3.5*

1.03

-61

153

36.7

80.14

3.3*
2.4*
5.3*

1.19
1.13
0.89

18.55
11.8
-114.1

189
101.1
78.5

46.7
2.2
24.5

84.17
96.16
52.11

56.96 (5)17.31 (7)

22.0
19.0
4.9*

0.89

-97.8

64.7

32.7

36.07

49.15 (7)54.00 (5)150.35 (3)135.91 (3)123.87 (3)73.78 (3)67.57 (5)25.37 (5)137.86 (1)129.24 (3)128.33 (3)125.49 (3)20.4 (7)164.2 (4)116.6 (4)126.3 (4)61.5 (4x5)

21.4
27.2
27.5
24.5
25.0

1.5*
5.0*

1.35
1.05

-95
-41.6

39.75
115.2-115.3

8.9
12.4

86.95
84.13

2.4-2.5

1.62
0.99

-44
-108.5

146.5
66

8.20
7.6

169.86
80.16

0.4*

0.94

-95

110.6

2.4

100.19

11.5

1.49

-15.4

72.4

115.03

5.0*

1.41

-43.5

74.05

103.06

13

JHD
(Hz)

2.3

2(9)

1

21.0
24.3
32.0
19.0
NA

22.2
20.2
23.0
24.0
24.0
19.0

22.0

M.J. O’Neil, P.E. Heckelman, C.B. Koch, K.J. Roman, The Merck Index, an Encyclopedia of Chemicals, Drugs, and Biologicals – Fourteenth Edition, Merck Co., Inc.
Whitehouse Station, NJ 2006.
�	
The 1H spectra of the residual protons and 13C
spectra were obtained on a Varian Gemini 200
spectrometer at 295°K. The NMR solvents used
to acquire these spectra contain a maximum of
0.05% and 1.0% TMS (v / v) respectively. Since
deuterium has a spin of 1, triplets arising fromcoupling to deuterium have the intensity ratio of
1:1:1. “m” denotes a broad peak with some finestructures. It should be noted that chemical shifts
can be dependent on solvent, concentration andtemperature.

�	Approximate values only; may vary with pH,
concentration, and temperature.
�	Melting and boiling points are those of thecorresponding unlabeled solvent (except for D2O).
These temperature limits can be used as a guide to
determine the useful liquid range of the solvents.
Information gathered from the Merck Index –Fourteenth Edition.

* HOD Peaks – NMR spectra of “neat” deuterated­solvent always exhibit a peak due to H2O in addition
to the residual solvent peak. When the exchange
rate between H2O and HOD is slow on the NMR
timescale the water peak appears as two peaks,a singlet ­corresponding to H2O and a 1:1:1 triplet
corresponding to HOD.

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Cambridge Isotope Laboratories, Inc.

| Stable Isotopes for Biomolecular NMR

Application Notes Summaries
APPLICATION NOTE 15

Cambridge Isotope Laboratories, Inc.
isotope.com

Top Ten Tips for Producing
C/ 15N Protein in AbundanceApplication Note 15

13

Deborah A. Berthold, Victoria J. Jeisy, Terry L. Sasser, John J. Shea,
Heather L. Frericks, Gautam Shah, and Chad M. Rienstra
Departments of Chemistry and Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801 USA
What could be easier than overexpressing an E. coli protein in
E. coli? You don’t have to be an old hand at protein expression
to know that this can often be more difficult than it sounds.
We tested our skills recently with DsbA, a 20 kDa protein thatcatalyzes disulfide bond formation in the E. coli periplasm.
The wildtype DsbA expressed well in LB medium and also in a
BioExpress®-supplemented 13C/ 15N-labeling medium. Likewise, theDsbA C33S mutant expressed well in LB. But when we first tried
to label C33S, our luck ran out – we saw no expression at all.
Today we are producing 13C/ 15N DsbA C33S at a yield of 100 mg
per liter. Here are our top ten tips for expressing recalcitrant proteins.
10. Stop the leaks. Leaky expression (i.e. expression in theabsence of inducer) of a “toxic” protein or even a “less thanhealthful” mutant protein can slow cell growth, resulting in a
suboptimal level of expression. In addition, because the half-lifeof ampicillin in a dense culture is less than 30 minutes, at laterstages of growth there is a loss of selection for cells with ampicillinresistant expression plasmids. So, any selection pressure stemmingfrom leaky expression would give advantage to cells that have losttheir expression plasmids. At best, the portion of plasmid-free cells
in the culture at the time of induction are taking up 13C-labeled
glucose without contributing to expression. At worst, leaky
expression slows cell growth to the point that there is no
growth at all in the labeling medium.
Promoters show variation in their leakiness, so use of a highly
regulated promoter, such as the T7 promoter of the pET vector
system, may be the solution. In our case, DsbA is on a high-copy
plasmid (pUC119) and is expressed from a lac promoter (Kisigami,
et al.,1995). In addition to this promoter being well-known for its
leakiness, in optimizing the expression of the wildtype DsbA, we
had changed E. coli strains. The original strain, E. coli M15/pREP4,
contained additional copies of the lac repressor on pREP4; ourpreferred strain, E. coli C43(DE3), had no additional repressor otherthan the copy in the host genome. It was likely that the repressor
binding sites on our expression plasmid titrated out all the copiesof the repressor in the cell, leaving some promoters unrepressed.
Our response: we put the compatible pREP4 plasmid into our E. coli
C43(DE3) so that the cell would produce enough repressor for all
the plasmid binding sites. Another solution might be to clone lacI q(a constitutive lac repressor) directly onto the expression plasmid;
this is found, for example, in Qiagen’s QE80 series of His-tag
expression vectors.
9. Slow down the train. For high levels of protein expression,
the rate of transcription needs to be coupled to that of translation,
which in turn needs to be coupled to any essential co- or posttranslational events, such as folding, cofactor binding or membrane
insertion. When transcription outstrips translation, loss of cell
viability can occur (along with the destruction of ribosomal RNA
and induction of proteases; see Dong, et al., 1995). There areseveral methods to tweak the rates of cellular metabolism to
try to bring transcription, translation and post-translational
processing in line. One can change promoters (Makrides, 1996;
Baneyx, 1999). Strong promoters, such as T7, can be replacedwith weaker promoters (arabinose, T5, tac). One can change
cell lines. Two E. coli strains particularly suited for expression
optimization, C41(DE3) and C43(DE3), were originally obtained
from a selection for mutations that overcame lethality associatedwith overexpression from a T7 promoter (Miroux and Walker, 1996).
These two strains also show an increase in plasmid stability relative
to their parent BL21(DE3) strain (Dumon-Seignovert, et al., 2004).
And, perhaps most easily, one can change the temperature during
expression. For DsbA, the interplay between growth temperatureand E. coli strain can be seen in Figure 1. Using BL21(DE3),
Continued ➤Cambridge Isotope Laboratories, Inc.
North America: 1.800.322.1174 cilsales@isotope.com | International: +1.978.749.8000 intlsales@isotope.com | fax: 1.978.749.2768 | isotope.com

Top Ten Tips for Producing 13C, 15N Protein in AbundanceDeborah A. Berthold, Victoria J. Jeisy, Terry L. Sasser, John J. Shea, Heather L. Frericks, Gautam Shah, andChad M. Rienstra

What could be easier than overexpressing an E. coli protein in E. coli? You don’t have to be an old hand at protein expression to knowthat this can often be more difficult than it sounds. We tested our skills recently with DsbA, a 20 kDa protein that catalyzes disulfide bondformation in the E. coli periplasm. The wildtype DsbA expressed well in LB medium and also in a BioExpress®-supplemented 13C, 15N-labelingmedium. Likewise, the DsbA C33S mutant expressed well in LB. But when we first tried to label C33S, our luck ran out – we saw noexpression at all. Today we are producing 13C, 15N DsbA C33S at a yield of 100 mg per liter. Here are our top ten tips for expressing
recalcitrant proteins. Read more of Application Note 15 at isotope.com.
APPLICATION NOTE 20

Cambridge Isotope Laboratories, Inc.
isotope.com

Effective Site-Specific Isotopic Labeling(15N/ 13C Glycine; 15N/ 13C Phenylalanine;15
N/ 13C Tryptophan) Expression
Optimization Using BioExpress®
2000 Media

Application Note 20

Kenneth C. Bonanno
Vertex Pharmaceuticals, Inc., 130 Waverly Street, Cambridge, MA 02139 USA

Recombinant genes have been expressed in a variety of cellular
systems for decades to generate protein reagents that are thepotential targets for new small-molecule drugs. As these targets
become more complex, researchers have developed innovative
methods to study the structure of these proteins and theinteractions with potential drugs. Cambridge Isotope Laboratories,
Inc.’s (CIL) BioExpress® 2000 media incorporates isotope-labeledamino acids into recombinant protein expressed in baculovirusinfected (BV) insect cells to assist NMR structural studies (Strauss,
et al., 2006). To use this labeled media most efficiently, expression
conditions must be optimized at a small scale prior to production.
The goal of this study was to maximize the expression ofa histidine-tagged, human serine/threonine kinase in the
Spodoptera frugiperda (SF9) cell line with site-specificisotopic labeling (15N/ 13C-labeled glycine; 15N/ 13C-labeledphenylalanine; 15N-labeled tryptophan) in BioExpress 2000
media (CIL catalog no. CGM-2000-Custom). The target enzyme
was expressed in BV-infected insect cells cultured in various small
volume (2 mL) conditions. Expression was optimized by adapting
a previously designed high-throughput small-scale expression
protocol (Chambers, et al., 2004) and incorporating Designof Experiment (DOE, Fisher, RA, 1925).
Three conditions most widely optimized for insect cell
expression are:• multiplicity of infection (MOI)• harvest time (hours post-infection – HPI• cell density at infection (cells/mL)
A matrix of these variables was explored in this experiment todetermine the maximum protein yield, measured in mg/L.

A Box-Behnken response surface model (Box & Behnken, 1960)
was applied in the experimental design using three equally spaced
factors (low, medium, high) for each of the three conditions (MOI,
HPI, cell density) in order to study the quantitative response, protein
yield. This model provides 13 conditions, including a centerpointwith replicates. DOE allows the exploration of many variables via
a limited number of experimental conditions using a statisticallysignificant model produced by the experimental data. (Chambers
and Swalley, 2008).
Cells were grown in 24-well block microplates (Whatman plc, UK)
and infected with BV at cell density and MOI specified by the
Box-Behnken design. Cell cultures were pelleted and frozen upon
harvest. Cell lysates were solubilized, and the target protein was
purified using nickel magnetic agarose beads (Sigma-Aldrich, MO).
Protein yield was determined using a dot blot assay with ananti-histidine primary antibody (BD BioSciences, NJ) and a goatanti-mouse secondary antibody that fluoresces at the 680 nm
wavelength (LI-COR Biosciences, NE) using the Odyssey® Infrared
Imaging System and the analysis software provided (LI-COR
Biosciences, NE) (Figure 1).
The expression data was analyzed with statistical software(JMP™ v6.0, NC) to determine if the data sufficiently fit themodel (Figure 2). The Box-Behnken model was verified since yielddata provided adequate fit (R2 = 80%) and there were significant
regression factors in the model (pyield < 0.001). Additionally, thep-value for the lack of fit test is not significant (p > 0.05). The data
predicted optimal conditions for the large-scale expression to
generate a yield of 1.3 ± 0.1 mg /L, a 20% improvement from thebest condition in the screen. As the case with many experiments
incorporating DOE, the optimal condition from the model (MOI = 5,Continued ➤Cambridge Isotope Laboratories, Inc.
North America: 1.800.322.1174 cilsales@isotope.com | International: +1.978.749.8000 intlsales@isotope.com | fax: 1.978.749.2768 | isotope.com

Effective Site-Specific Isotopic Labeling (15N, 13C Glycine; 15N, 13C Phenylalanine;
15
N Tryptophan) Expression Optimization Using BioExpress® 2000 Media
Kenneth C. Bonanno

Recombinant genes have been expressed in a variety of cellular systems for decades to generate protein reagents that are the potential
targets for new small-molecule drugs. As these targets become more complex, researchers have developed innovative methods to study the
structure of these proteins and the interactions with potential drugs. Cambridge Isotope Laboratories, Inc.’s (CIL) BioExpress® 2000 media
incorporates isotope-labeled amino acids into recombinant protein expressed in baculovirus-infected insect cells to assist NMR structural
studies (Strauss, et al., 2006). To use this labeled media most efficiently, expression conditions must be optimized at a small scale prior
to production. Read more of Application Note 20 at isotope.com.
APPLICATION NOTE 22

Cambridge Isotope Laboratories, Inc.
isotope.com[2,3-13C]-Labeled Aromatic Residues as a
Means to Improving Signal Intensities
and Kick-Starting the Assignment of
Membrane Proteins by Solid-State
MAS-NMR

Application Note 22

Matthias Hiller, Victoria A. Higman, Stefan Jehle, Barth-Jan van Rossum,
Werner Kühlbrandt, and Hartmut Oschkinat
Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany
and Max-Planck-Institut für Biophysik, Max-von-Laue-Str. 3, 60438 Frankfurt am Main, Germany
Over the past few years solid-state MAS-NMR has rapidly beendeveloping into a structure determination technique for biologicalmacromolecules.1-6 Its advantages include the ability to study
membrane proteins in their native lipid environment,7-10 as wellas making possible the study of nonsoluble or noncrystallisable
protein states, such as amyloid fibrils.11-15 However, a prerequisite
for structure determination is a high level of resonance assignment.
There are numerous examples of small- and medium-sized proteinsfor which this has been possible,4,16-20 but for large membraneproteins, such as the 281-residue outer membrane protein G(OmpG)21-22 resonance assignment still remains a challenge.
Signal overlap is an obvious problem, but fast longitudinal andtransverse relaxation rates also contribute toward lower signal/
noise ratios. Furthermore, membrane proteins can reduce the
Q-factor of the coil, and the experimentalist is then left with thedifficult task of balancing increased decoupling powers against
possible sample heating which could lead to sample degradation.
These problems result in lower signal intensities in proton-driven
spin diffusion (PDSD) spectra; however, they can be addressed using
several different strategies, such as by improving coil design,23-25 bydeveloping spectral editing pulse sequences,26,27 or by using novel
labeling strategies.28,29 It is this last approach, using a novel labelingstrategy, which is presented here in this application note.
It has been shown that samples produced using [1,3-13C]- and[2-13C]-labeled glycerol in the bacterial growth medium give rise tospectra with improved cross-relaxation properties and which provevery helpful at the restraint generation stage.1 But assignment using
solely these samples is not possible, since the C’, Cα and Cβ atoms
are never simultaneously labeled. An alternative approach would be
to use [1,2,3-13C]-, [1,2-13C]- and/or [2,3-13C]-labeled amino-acids.
This would retain the favorable cross-relaxation properties of theglycerol samples by keeping the number of consecutively bonded
labeled carbon atoms low, but would provide a more useful setof labels for assignment. The two former labeling patterns caneasily be generated with uniformly labeled short amino acids suchas alanine, serine, cysteine, or glycine. Additional incorporationof other amino acids with [2,3-13C] labeling would increase thenumber of detectable amino acids but reduce the overlap in
NCO-type spectra,30 thus providing scope for many unambiguous
sequential assignments from these spectra. When considering
which amino acid types to select for [2,3-13C] labeling, it is worth
noting that Cβ signals from aromatic amino acids are often very
weak or not visible at all. [2,3-13C] labeling aromatic amino acids
would reduce J-couplings at the Cα and Cβ positions, as well as
ensure that the aromatic ring (which undergoes fast relaxation)
does not draw away magnetization and effectively act as amagnetization “sink.” The [2,3-13C] labeling would then not
only aid assignment, but also improve the spectral quality andidentification of aromatic spin systems.
This application note describes the use of a novel labeling strategy
to increase the spectral quality and level of resonance assignment
in MAS-NMR spectra of the biological macromolecule OmpG.
Based on the protein’s sequence, a labeling scheme of fully[15N,13C]-labeled Ala and Gly and [2,3-13C,15N]-labeled Tyr and
Phe was selected. The sample (hereafter referred to as OmpGGAFY) was prepared using established protocols and incorporating[2,3-13C,15N]-Tyr (CIL #CNLM-7610), [2,3-13C, 15N]-Phe (CIL #CNLM7611), [U-13C3,15N]-Ala (CIL #CNLM-534) and [U-13C2,15N]-Gly(CIL #CNLM-1673).

Continued ➤Cambridge Isotope Laboratories, Inc.
North America: 1.800.322.1174 cilsales@isotope.com | International: +1.978.749.8000 intlsales@isotope.com | fax: 1.978.749.2768 | isotope.com[2,3-13C]-Labeled Aromatic Residues as a Means to Improving Signal Intensities andKick-Starting the Assignment of Membrane Proteins by Solid-State MAS-NMR
Matthias Hiller, Victoria A. Higman, Stefan Jehle, Barth-Jan van Rossum, Werner Kühlbrandt and Hartmut Oschkinat

Over the past few years solid-state MAS-NMR has rapidly been developing into a structure determination technique for biologicalmacromolecules.1-6 Its advantages include the ability to study membrane proteins in their native lipid environment,7-10 as well as making
possible the study of non-soluble or non-crystallisable protein states, such as amyloid fibrils.11-15 However, a prerequisite for structure
determination is a high level of resonance assignment. There are numerous examples of small- and medium-sized proteins for which this has
been possible,4,16-20 but for large membrane proteins, such as the 281-residue outer membrane protein G (OmpG)21-22 resonance assignment
still remains a challenge. Signal overlap is an obvious problem, but fast longitudinal and transverse relaxation rates also contribute towards
lower signal/noise ratios. Furthermore, membrane proteins can reduce the Q-factor of the coil, and the experimentalist is then left with thedifficult task of balancing increased decoupling powers against possible sample heating which could lead to sample degradation. These
problems result in lower signal intensities in proton-driven spin diffusion (PDSD) spectra, however, they can be addressed using several
different strategies, such as by improving coil design,23-25 by developing spectral editing pulse sequences,26-27 or by using novel labelingstrategies.28-29 It is this last approach, using a novel labeling strategy, which is presented here in this application note. Read more ofApplication Note 22 at isotope.com.

Please visit isotope.com for additional bioNMR application notes.

58

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Application Notes Summaries
APPLICATION NOTE 25

Cambridge Isotope Laboratories, Inc.
isotope.com

Isotope Labeling of Alanine MethylGroups on a Deuterated Backgroundfor NMR Studies of High-MolecularWeight Proteins

Application Note 25

Chenyun Guo, Raquel Godoy-Ruiz, and Vitali Tugarinov
Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742 USA

State-of-the-art isotope-labeling schemes commonly employedfor NMR investigations of high-molecular-weight proteins utilize
selective incorporation of protons and 13C isotopes into methylgroups of Ileδ1, Leuδ and Valγ side chains in a highly deuterated
environment (commonly referred to as “ILV labeling”) providing
a large number of high-quality probes for NMR studies of protein
structure and dynamics. Robust ILV labeling methods1,2 and strategic
location of ILV side chains in the hydrophobic cores of protein
structures3,4 have turned ILV labeling into an indispensable tool for
NMR studies of large proteins and macromolecular assemblies.5
More than a decade ago, Gardner & Kay pioneered a technique
for selective incorporation of protonated Ileδ1 methyl positions
into protein molecules.6 Subsequently, selective methyl labeling/
protonation methods have been refined and extended to Leuδ andValγ methyl sites by the Kay group.7,8 The introduction of methyltransverse relaxation optimized spectroscopy9 (methyl-TROSY) has
stimulated the development of improved labeling schemes.10,11
Selective labeling of ILV methyl sites in large proteins on a
deuterated background in synergy with methyl-TROSY techniques
have had a significant impact on NMR studies of structure anddynamics of large protein assemblies up to ~800 kDa in molecularweight.12-15 Nevertheless, the availability of only three ILV probes
oftentimes presents a serious limitation for structural and dynamics
studies of large proteins. Recent advances in methyl isotope
labeling have focused on the extension of the ILV labelingmethodology to methyl positions of alanines (Alaβ).

Methyl groups of alanines serve as an attractive extension of the
ILV labeling methodology. Ala methyls are located in close proximity
to the backbone; their flexibility is therefore reduced compared toother methyl-bearing side chains in proteins. Ala is one of the mostfrequently encountered residues in protein hydrophobic cores andat molecular interfaces.16 Recently, several reports have focusedupon extending the ILV methodology with (selective) labelingof Alaβ methyl positions in large proteins. Isaacson et al. haveintroduced a labeling strategy for selective incorporation of
13
CH3 groups into alanine residues on a deuterated background.17
Specific 13C labeling and protonation of Alaβ methyls to a level of
95% with minimal background labeling (<1%) in minimal D2Obased bacterial medium supplemented with large amounts of
selectively 13C-labeled α-deuterated alanine and co-additionof three deuterated compounds: (i) α-ketoisovalerate-D7, (ii)succinate-D4 and (iii) L-isoleucine-D10 has been reported by
Boisbouvier and coworkers.18 This labeling protocol has beenclosely followed for production of {2H; Alaβ-[13CH3]}-labeled malate
synthase G19,20 (MSG) – an 82-kDa enzyme containing 73 methylgroups. Alanine is the most abundant residue in MSG comprising
10.1% of the total amino-acid content. Figure 1 shows the methylTROSY 1H-13C correlation map of MSG prepared using the protocol
described in more detail in Appendix 1.
To maximize the number of available methyl probes, it is clearlyadvantageous to combine selective labeling of Ala positions
with ILV methyl labeling.21 The use of selectively 13CH3-labeled

Continued ➤Cambridge Isotope Laboratories, Inc.
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Isotope Labeling of Alanine Methyl Groups on a Deuterated Background for NMR
Studies of High-Molecular-Weight Proteins
Chenyun Guo, Raquel Godoy-Ruiz and Vitali Tugarinov

The state-of-the-art isotope-labeling schemes commonly employed for NMR investigations of high-molecular-weight proteins utilize
selective incorporation of protons and 13C isotopes into methyl groups of Ileδ1, Leuδ and Valγ side chains in a highly deuterated environment(commonly referred to as “ILV labeling”) providing a large number of high-quality probes for NMR studies of protein structure and dynamics.
Robust ILV labeling methods1,2 and strategic location of ILV side-chains in the hydrophobic cores of protein structures3,4 have turned ILV
labeling into an indispensable tool for NMR studies of large proteins and macromolecular assemblies.5 More than a decade ago, Gardner and
Kay pioneered a technique for selective incorporation of protonated Ileδ1 methyl positions into protein molecules.6 Subsequently, selective
methyl labeling/protonation methods have been refined and extended to Leuδ and Valγ methyl sites by the Kay group.7,8 The introductionof methyl transverse relaxation optimized spectroscopy9 (methyl-TROSY) has stimulated the development of improved labeling schemes.10,11
Selective labeling of ILV methyl sites in large proteins on a deuterated background in synergy with methyl-TROSY techniques have hada significant impact on NMR studies of structure and dynamics of large protein assemblies up to ~800 kDa in molecular weight.12-15
Nevertheless, the availability of only three ILV probes oftentimes presents a serious limitation for structural and dynamics studies of large
proteins. Recent advances in methyl isotope labeling have focused on the extension of the ILV labeling methodology to methyl positionsof alanines (Alaβ). Read more of Application Note 25 at isotope.com.
APPLICATION NOTE 39

Cambridge Isotope Laboratories, Inc.
isotope.com

Production of U-[2H], Thr-γ2[13CH3]
Labeled Proteins for Methyl-TROSY
NMR
Lewis Kay, PhD
Departments of Molecular Genetics, Biochemistry and Chemistry
University of Toronto, Ontario, Canada

Isotope labeling has revolutionized the utility of biomolecular NMR
spectroscopy, allowing the exploration of molecular interactions
with high sensitivity and resolution.1,2 Many different strategies
are available, along with a wide array of NMR experiments thatare optimized for the different labeling approaches. One schemethat has been shown to be particularly effective in studies of highmolecular-weight proteins involves labeling methyl groups as 13CH3
in an otherwise highly deuterated background3,4,5,6,7 and exploitinga methyl-TROSY effect8 that generates high-quality spectra.
Applications to date have focused to a large extent on Ile, Leu
and Val methyl probes,9 as the precursors for these residues
are commercially available and very easy to use. More recently,
however, studies utilizing Met10,11 and Ala12 methyl groups havealso emerged along with approaches for introducing methyls
into key positions in the protein of choice.
It is of significant interest to extend the methyl-labelingmethodology to include Thr residues, as Thr has a muchhigher propensity for surface exposure than the other methylcontaining residues.13,14 As such, Thr is often found at key
molecular interfaces, including those involving in binding nucleic
acids. Our laboratory has developed a biosynthetic strategy15 thatstarts with 13C-formaldehyde, natural-abundance pyruvate and D2O,
along with five enzymes that are necessary for the conversion toU-[2H], Thr-γ2[13CH3]. The development of a synthetic scheme by
Cambridge Isotope Laboratories, Inc. (CIL) and the commercial
availability of this product is a welcome addition, since a fiveenzyme synthesis is usually something that NMR spectroscopists
like to avoid!

Production of U-[2H], Thr-γ2[13CH3]-labeled samples is straightforward. As described previously,15 the addition of Thr to growth
media does lead to labeling at both Thr γ2 and Ile δ1 positions, as
is expected since Thr is a precursor of Ile. Because cross peaks for Ile
residues fall in an isolated region of the 13C, 1H correlation map andare among the most well resolved of all methyl types, we prefer toinclude Ile labeling in all of our Thr samples (and often also Leu, Val).
In this regard, we recommend using 50 mg/L labeled U-[2H], Thrγ2[13CH3], 50 mg/L labeled α-ketobutyrate (13CH3CD2COCOONa)
and 100 mg/L d5-glycine as an optimal combination for productionof highly deuterated proteins labeled with 13CH3 at Thr and Ile (δ1)
methyl positions. Further details can be found in reference 15.
All of these compounds can be purchased from CIL.

Application Note 39

Thr plays a critical role in the mechanism of function of a numberof important enzymes and in several eukaryotic signaling complexes
where biological activity is regulated through phosphorylation.
Our research using U-[2H], Thr-γ2[13CH3] to probe the catalytic
mechanism of a class of barrel-shaped proteases that includes boththe proteasome16 and HslV17 has more than convinced us of theutility of this product, and one can easily imagine other systems
where it would be equally useful. As an example of the importanceof using precursors that are deuterated at all positions with the
exception of the methyl groups, Figure 1 illustrates comparative
spectra recorded on proteasome samples (670 kDa) that are highly
deuterated and prepared labeled through the addition of either
U-[13C, 1H]-Thr (a) or U-[2H], Thr-γ2[13CH3] (b). The difference isapparent, and the signal from the methyl group of the important
catalytic residue, Thr1, is only visible in the spectrum of the protein
prepared with the 2H-Thr precursor.16

Production of U-[2H], Thr-γ2[13CH3] Labeled Proteins for Methyl-TROSY NMR

Continued ➤Cambridge Isotope Laboratories, Inc.
North America: 1.800.322.1174 cilsales@isotope.com | International: +1.978.749.8000 intlsales@isotope.com | fax: 1.978.749.2768 | isotope.com

Lewis Kay, PhD

Isotope labeling has revolutionized the utility of biomolecular NMR spectroscopy, allowing the exploration of molecular interactions withhigh sensitivity and resolution.1,2 Many different strategies are available, along with a wide array of NMR experiments that are optimized forthe different labeling approaches. One scheme that has been shown to be particularly effective in studies of high-molecular-weight proteins
involves labeling methyl groups as 13CH3 in an otherwise highly deuterated background3,4,5,6,7 and exploiting a methyl-TROSY effect8 thatgenerates high-quality spectra. Applications to date have focused to a large extent on Ile, Leu and Val methyl probes,9 as the precursors forthese residues are commercially available and very easy to use. More recently, however, studies utilizing Met10,11 and Ala12 methyl groups
have also emerged along with approaches for introducing methyls into key positions in the protein of choice. Read more of Application
Note 39 at isotope.com.
APPLICATION NOTE 48

Cambridge Isotope Laboratories, Inc.
isotope.com

Stereospecific Leu/ Val Methyl Labeling:
An Important Technology for NMR
Studies of High-Molecular-Weight
Complexes

Application Note 48

Rui Huang1 and Lewis E. Kay1,2
1. Departments of Molecular Genetics, Biochemistry and Chemistry, University of Toronto, Toronto, Ontario, M5S 1A8 Canada
2. Hospital for Sick Children, Program in Molecular Medicine, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada

The development of modern biomolecular solution NMR
spectroscopy has paralleled innovations in labeling technologies.
For example, early advances in heteronuclear double- and tripleresonance NMR spectroscopy were closely coupled to theemergence of methodologies for the effective uniform labeling of
biomolecules with 15N and/or 13C.1,2 Despite arguments that uniform13
C (U-13C) labeling would be prohibitively expensive, the highdemand for U-13C-labeled precursors has significantly lowered costs,
and the preparation of biomolecules with 13C incorporation is mostoften the approach of choice for NMR studies.3 As the size of thesystem studied increases, both the sensitivity and the resolution of
the resulting spectra suffer, and this has led to the development of
amide-TROSY-based experiments4 that are significantly improved
through the use of perdeuteration.5 A powerful labeling scheme forstudies of proteins over a wide range of molecular masses, but, inparticular, for systems with aggregate masses in excess of 100 kDa
involves 13CH3-labeling in a highly deuterated background.6 Initial
schemes focused on Ile (d1), Leu and Val methyl groups, where for
Leu and Val only one of the isopropyl methyls is 13CH3 (the other is
12CD3), but more recently precursors for the other methyl positions
have become commercially available7-11 so that it is possible to label
the methyl group of choice. Once again, the availability of precursors
stimulated the development of new NMR experiments, in this casethose exploiting a methyl-TROSY effect,12 which has resulted inapplications involving protein complexes in excess of 1 MDa.13
The continued success of NMR studies of molecular machines overthe past years has led to the study of increasingly complex targets.
For example, initial studies by our laboratory focused on the 670
kDa proteasome core particle,14 that is comprised of 14 copies ofa and b-subunits, each with a molecular mass of approximately
25 kDa. The relatively small size of each protomer, albeit within thecontext of a large complex, meant that resolution was not limiting
and that experiments could be performed using samples where
methyl groups of Leu and Val were not stereospecifically 13CH3
labeled, with one isopropyl methyl NMR-active in each Leu and Val.
As the size of the individual protomers of the complex increases,
spectral resolution is challenged as a pair of signals is measured foreach isopropyl group. Boisbouvier and coworkers have developed
an elegant approach that significantly addresses this limitation byintroducing a synthetic route for the production of specificallymethyl-labeled acetolactate, subsequently converted into either
proR- or proS-13CH3 a-ketoisovalerate in vivo that then produces
Leu and Val with 13CH3 labeling confined to a single prochiralmethyl15 (Figure 1A). In addition, to simplifying the resulting NMR
spectrum, the sensitivity of cross-peaks is increased two-fold relative
to samples prepared with the racemic pro-R/pro-S 13CH3,12CD3isovalerate mixture as all of the label is focused on a single position
rather than a 50%-50% split. This is an important consideration inapplications involving samples that are concentration limited, and,in general in studies of very high-molecular-weight complexes.
Our recent work on the proteostasis protein p9716 provides anexample of the utility of the stereospecific biosynthetic labelingscheme of Boisbouvier.15 Here we focused on a 320 kDa hexamericfragment of p97, with each of the protomers comprising residues
1-480 of the 540 kDa intact protein. A series of p97 disease
mutants had been characterized biochemically prior to ourstudy, but little structural information was available that provided
a firm understanding of the relation between the mutants and
p97 malfunction; indeed X-ray-derived structures of wild-type
and mutated proteins were very similar. Figure 1B shows the
Leu/ Val region of the 13C-1H HMQC spectrum recorded on asample of highly deuterated, Ile Cd1-13CH3, proR Leu,Val Cd,Cg-13CH3,

Continued ➤Cambridge Isotope Laboratories, Inc.
North America: 1.800.322.1174 cilsales@isotope.com | International: +1.978.749.8000 intlsales@isotope.com | fax: 1.978.749.2768 | isotope.com

Stereospecific Leu/ Val Methyl Labeling: An Important Technology for NMR Studies
of High-Molecular-Weight Complexes
Rui Huang and Lewis E. Kay

The development of modern biomolecular solution NMR spectroscopy has paralleled innovations in labeling technologies. For example, earlyadvances in heteronuclear double- and triple-resonance NMR spectroscopy were closely coupled to the emergence of methodologies for theeffective uniform labeling of biomolecules with 15N and/or 13C.1,2 Despite arguments that uniform 13C (13C) labeling would be prohibitively
expensive, the high demand for 13C-labeled precursors has significantly lowered costs, and the preparation of biomolecules with 13C
incorporation is most often the approach of choice for NMR studies.3 As the size of the system studied increases, both the sensitivity andthe resolution of the resulting spectra suffer, and this has led to the development of amide-TROSY-based experiments4 that are significantly
improved through the use of perdeuteration.5 A powerful labeling scheme for studies of proteins over a wide range of molecular masses,
but, in particular, for systems with aggregate masses in excess of 100 kDa involves 13CH3-labeling in a highly deuterated background.6 Initial
schemes focused on Ile (d1), Leu and Val methyl groups, where for Leu and Val only one of the isopropyl methyls is 13CH3 (the other is 12CD3),
but more recently precursors for the other methyl positions have become commercially available7-11 so that it is possible to label the methylgroup of choice. Once again, the availability of precursors stimulated the development of new NMR experiments, in this case those exploitinga methyl-TROSY effect,12 which has resulted in applications involving protein complexes in excess of 1 MDa.13 Read more of Application
Note 48 at isotope.com.

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BIONMR_CATALOG (1/15/20)
Supersedes all previously published literature