THE NEWSLETTER OF ACTIVE MOTIF — SEPTEMBER 2004 • VOLUME 5 • NUMBER 3

ChIP-IT™ – Chromatin Immunoprecipitation Made Easy
kit includes positive and negative control
antibodies and PCR primers that help you
interpret your results. With ChIP-IT you
get a complete package of high-quality,
optimized reagents, ensuring that you get
the best results possible, while saving you
both time and money.
The power of ChIP
Transcriptional regulation is one of themain methods used by cells to control
gene expression. Traditionally, researchers
have analyzed transcription factors andcontinued on page 4

Phospho-specific ELISAs for Bad, c-Src, FAK, FKHR,
GSK3β, JAK & PI3KFACE™ (Fast Activated Cell-based ELISA)
Kits offer a better alternative to Western
blots and Sandwich ELISAs for the studyof protein phosphorylation because thecell-based format of FACE allows you to
grow, fix and assay your cells all in the sameplate. This keeps protein modifications to aminimum, enabling you to assess the exact
protein state within the cell at your chosen
time point. Now, Active Motif’s popular
FACE Kits are available for monitoring boththe phosphorylated and native forms ofBad, c-Src, FAK, FKHR, GSK3β, JAK1 and PI3
Kinase p85. And, for added convenience,
FACE Kits are offered in both colorimetricand ultra-sensitive chemiluminescentformats, giving you the flexibility to choose
the kit that best fits your needs and yourin-house equipment. Studying phosphoproteins has never been so simple.

The FACE method
In FACE, cells are cultured in the provided
96-well plates and stimulated to induce thepathway of interest (Figure 2, pg. 6). The cells
are then rapidly fixed with formaldehyde
to preserve protein modifications, including
phosphorylation. Each well is then incubatedwith a primary antibody specific for thephospho-protein of interest. This is followed
by secondary HRP-conjugated antibody
and developing reagent, which provides asensitive colorimetric or chemiluminescentreadout. FACE Kits come complete with everything needed to perform 96 assays usinga phospho-antibody that is directed against
the phosphorylated form of the protein ofinterest, as well as 96 assays using a total
antibody. Using two different antibodies
enables simultaneous analysis of both thenative and activated protein levels (Figure 1).

1

ChIP-IT™ – Chromatin
Immunoprecipitation Made Easy

1

Phospho-specific ELISAs for
Bad, c-Src, FAK, FKHR, GSK3β,
JAK & PI3K

2

Sensitive, Non-radioactive
Method for Studying Transcription Factor Activation

3

Chariot™ – A Fast, Convenient
Alternative to DNA Transfection

5

Improved Gene Silencing
Through Higher Specificity
Binding

7

Faster, More Accurate Measurement of NO Production

8

Efficient, Affordable
Competent E. coli

100000Uninduced
FBS Induced

80000

RLU

Classically, chromatin immunoprecipitation(ChIP) has been performed using reagents
prepared “in house”, or with commercial
kits that do not contain everything thatis needed to complete the experiment.
This can be time consuming and frustrating, particularly as there are no guarantees
that the separate components will work
well together. To overcome this problem,
Active Motif has developed its ChIP-IT™ Kit.
ChIP-IT is a complete solution that contains
all of the reagents required for DNA shearing, “pull down” and purification. Plus, theIN THIS ISSUE

60000

40000

20000
0

Total-FKHR

Phospho-FKHR

Figure 1: Measurement of phosphorylated and total FKHR.
CHO-K1 cells were cultured in 96-well plates and serum-starved for 16
hours. Cells were then treated with 20% FBS for 30 minutes and fixed.
Total and phospho FKHR were each assayed in triplicate using thephospho and total FKHR antibodies included in the FACE FKHR Chemi
Kit. Data was plotted after correction for cell number (performed
through use of the kit’s Crystal Violet reagent).

continued on page 6

www.activemotif.com

Transcription Factor ELISAs

Sensitive, Non-radioactive Method for Studying Transcription Factor Activation
Active Motif’s TransAM™ Kits are ELISAbased assays* that simplify the study oftranscription factor activation in mammalian tissue and cell culture extracts.
The TransAM method is up to 100-foldmore sensitive than the gelshift techniqueand provides results in less than 5 hours.
TransAM Kits eliminate the use of radioactivity, while their flexible format makes it
possible to screen from 1 to 96 samples ina single experiment. And, unlike reporter
assays, TransAM Kits can be used to study
all sample types, including cell lines and tissues, giving you unsurpassed flexibility.
Simple, specific method
Each TransAM Kit includes a 96-well platein which multiple copies of a specificdouble-stranded oligonucleotide havebeen immobilized. When nuclear or wholecell extract is added to the wells of theplate, the transcription factor of interest
binds to its consensus-binding site on theoligonucleotide. A primary antibody thatis specific for the activated form of thetranscription factor is then added, followed
by incubation with a secondary HRP-conjugated antibody and developing reagent.
The colorimetric change is read using aspectrophotometer, providing a sensitive,
quantitative measurement of transcription
factor activation (Figure 1). The novel ELISA
format is readily adaptable to automatedhigh-throughput screening, which further
reduces the time and labor normally associated with quantifying levels of activatedtranscription factors.

Add nuclear
extract

Add primary
antibody

Add anti-IgG
HRP conjugate

1 hr.

1 hr.

Add developing
and stop solution

1 hr.

Cell extractcontaining activatedtranscription factor

Oligonucleotide
coated plate

Figure 1: Flowchart of the TransAM procedure.

Sensitive, quantitative assay
Because small changes in transcription
factor levels have a significant impact on
cellular function, it is vital to use a sensitive
assay. TransAM Kits are 10-fold more sensitive than gelshift assays, while TransAM
Chemi Kits provide 100-fold better sensitivity. And, because TransAM assays provide
quantitative results, you can subtract thelevel of endogenous factor present in youruntreated samples from your test samples,
further improving your results.
TransAM assay formats
TransAM Kits are offered in a variety offormats – individual kits assay single transcription factors, while Family Kits make itpossible to profile multiple members of atranscription factor family. And, TransAM
Chemi Kits utilize a luminometer to provide
the maximum sensitivity possible. Currently
available TransAM assays are listed below.

TransAM advantages• Colorimetric method provides
sensitive, quantifiable results• Eliminates the use of radioactivity andthe need to run and develop gels
• Results in less than 5 hours• Profile multiple members of a transcription factor family simultaneously• Ability to assay cell and tissue samples
Complete solution for activation assays
TransAM Kits will improve your transcription factor activation research because
they are faster, more sensitive and easier to
use than existing methods. Please visit our
website at www.activemotif.com/transam
to learn about the latest additions or to
download TransAM manuals and a list of
publications that cite the use of TransAM.
For a better method for studying transcription factor activation, try TransAM today.
* Technology covered under AAT-filed patents licensed to Active Motif.

TransAM Product Line
TransAM™ AP-1 Family

TransAM™ AML-3/Runx2

TransAM™ C/EBP α/βTransAM™ IRF-3

TransAM™ NFκB p65

TransAM™ GATA Family

TransAM™ AP-1 c-Fos

TransAM™ CREB & pCREB

TransAM™ MEF2

TransAM™ NFκB p65 Chemi**

TransAM™ HNF Family

TransAM™ AP-1 c-Jun

TransAM™ Elk-1

TransAM™ MyoD

TransAM™ Oct-4

TransAM™ IRF Family

TransAM™ AP-1 FosB

TransAM™ ER

TransAM™ NF-YA

TransAM™ p53

TransAM™ MAPK Family

TransAM™ AP-1 JunD

TransAM™ FKHR (FOXO1)TransAM™ NFATc1

TransAM™ PPARγTransAM™ NFκB Family

TransAM™ ATF-2

TransAM™ HIF-1

TransAM™ NFκB p50

TransAM™ Sp1 & Sp1/Sp3

TransAM™ STAT Family

TransAM™ c-Myc

TransAM™ HNF-1

TransAM™ NFκB p50 Chemi**

TransAM™ STAT3** TransAM Chemi Kits require the use of a luminometer.

2

Toll Free — 1 877 222 9543

SEPTEMBER 2004 • VOLUME 5 • NUMBER 3

Protein Transfection

Chariot™ – A Fast, Convenient Alternative to DNA Transfection
Despite their wide-spread use, DNAtransfection and gene expression aresuboptimal techniques for determining
what effects are caused by introducing
recombinant proteins into cells. Lipidbased transfection methods have long
been known to be cytotoxic to most celllines. This, coupled with overexpressionof the recombinant protein, can cause anumber of changes in the cell that are completely independent of the actions of theintroduced protein. Thus, it can be difficult
to determine if any observed changes arecaused by the recombinant protein or by
the transfection/expression method itself.
In addition, the need to wait days to weeks
after transfection to assay for the recombinant protein is not only inconvenient, it is awaste of both time and resources.
Keep in control
In stark contrast, Active Motif’s Chariot™ is
an innovative alternative that is fast, convenient and overcomes the limitations ofclassical DNA transfection. Chariot is a noncytotoxic peptide carrier that efficiently
transports biologically active proteins,
peptides and antibodies directly into cells
in less than two hours. Not only do you getyour results sooner, but you can control thetiming and amount of your protein that isdelivered. This makes Chariot the delivery
vehicle of choice for functional studies.
Fast, stable protein delivery
During a brief 30-minute incubation, Chariot
and the macromolecule of interest form astable, non-covalent complex. This complex
protects the macromolecule from degradation during delivery, which helps to preserve
its function. Upon addition to cells, thecomplex is rapidly internalized, where it dissociates. The Chariot peptide then localizes
to the cell nucleus where it is degraded,leaving the macromolecule fully functionaland free to proceed to its target organelle(Figure 1). By directly delivering the protein,
peptide or antibody, Chariot completely
bypasses the transcription-translation
process associated with gene expression,saving hours to days of time.

www.activemotif.com

Cell line or model

Cell type

Macromolecule delivered

3T3-L1

Mouse fibroblast

Antibody

80%

A549

Human lung carcinoma

Antibody

95%

Arabidopsis

Primary plant protoplasts

ProteinNot provided

COS-7

Monkey kidney

Protein, oligopeptide

80%

CV-1

Monkey kidney

Antibody

55%

HCA2

Human fibroblasts

Protein90%

HeLa

Human cervix carcinoma

Protein, peptide, antibody

95%

HMSC

Primary human mesenchymal stem cells

Peptide

80%

HS-68

Human foreskin fibroblastProtein, peptide, antibody

95%

IMR90

Human fibroblastProtein, peptide

80%

JurkatHuman T-cell leukemia

Protein, peptide, antibody

75%

Mono Mac 6

Primary human monocyte

Protein50%

Mouse hepatocytes

Primary liver

Protein95%

Mouse model (in vivo)

Alveolar wall tissue

Protein88%

Neural retina cells

Primary chicken

Protein80%

NIH/3T3

Mouse embryoProtein, peptide

80%

NRK

Normal rat kidney

Antibody

30-60%

PC-12

Rat pheochromocytoma

Protein, peptide

80%

Sensory neurons (DRG)

Primary chick

Antibody, peptide

80%

Sensory neurons (DRG)

Primary rat

Protein80%

Thyrocytes

Primary humanProtein

90%

WI-38

Human lung fibroblastProtein, peptide, antibody

95%

WISH

Human placenta carcinoma

Protein30%

Do more with Chariot
The ability to deliver functionally active
protein, peptides and antibodies into cells
makes it easy to perform studies that are
not possible using classical methods. Thereis an extensive list of publications on our
website describing such novel research, aswell as a complete list of delivery efficiencies provided by Chariot users (Table 1).

Delivery efficiency (%)

Association

Cell targeting

Membrane translocation

Decaging

To find out more about how Chariot can
improve your research, give us a call today
or send in the enclosed reply card.

Figure 1: Schematic model for translocation of the Chariot complex.
Figure generously provided by Dr. Gilles Divita, Biophysics Dept.,
CNRS, Montpellier, France.1

Product

FormatCatalog No.

Chariot™

25 rxns*
100 rxns

30025
30100

β-Galactosidase Staining Kit

75 rxns

35001

1. Deshayes et al. (2004) Biochemistry 43:1449-1457.
* A rxn is defined as sufficient reagent to deliver protein to cells in a 35 mm plate.

3Chromatin Immunoprecipitation

continued from page 1 — ChIP-IT™ – Chromatin Immunoprecipitation Made Easy
gene promoter activity using methods suchas reporter gene assays, EMSA, Western
blotting and, more recently, using DNA
microarrays. However, although thesemethods have led to significant advancesin the scientific understanding of transcription, there is still a need for improved
assays. Specifically, there is a need forbetter understanding of in vivo interactions
between DNA and transcription factors.
The ChIP method overcomes this limitation through a formaldehyde cross-linking
step, which “freezes” and preserves cellular
protein/DNA interactions.
The ChIP-IT method
In the ChIP-IT method, intact cells are fixedusing formaldehyde, which cross-links andtherefore preserves protein/DNA interactions. DNA is then sonicated into small,uniform fragments and the DNA/protein
complexes are immunoprecipitated using
antibody(ies) directed against the DNAbinding protein(s) of interest. Following
immunoprecipitation, the cross-linking isreversed and DNA fragments are screened
to determine which gene or groups of genes
had bound the protein of interest (Figure 1).
The keys to successful ChIP
Successful ChIP depends on a variety ofdifferent factors, including correct fixationof the DNA, shearing of the DNA to an
optimal length and possessing both anantibody that recognizes the exposed
motifs of fixed, DNA-bound protein as
well as a PCR primer pair that specifically
amplifies the region of interest. A variety of
specialized buffers, inhibitor cocktails andblocking reagents are required during thesesteps to minimize non-specific enrichment
and reduce background. Should any of yourreagents fail to work properly or not be
optimized for use with one another, yourChIP experiment may fail, wasting precious
time and resources. However, the ChIP-IT
Kit was designed to eliminate such worries.
All reagents in the kit are optimized for use
together and are rigorously QC’d to ensurethat you get the best results possible.

4

Cross-link protein to DNA inliving cells with formaldehyde

PCR
primers
Induction

TargetDNA

Break open cells
and shear DNA

Add pre-blocked
Protein G Agarose Beads

Add primary
antibody of interest

Negative
Control

Detect and quantify
precipitated DNA through
PCR and hybridization methods

Reverse cross-links andtreat with proteinase K

Immunoprecipitate to enrich
for fragments bound byprotein of interest

Figure 1: Schematic of chromatin immunoprecipitation. In ChIP, protein/DNA interactions are fixed, and the DNA is then sheared and precipitated using anantibody. After reversing the cross-links, the DNA is purified and then screened to determine which genes were bound by the protein of interest.

ChIP-IT advantages• Complete solution for ChIP – all critical reagents and controls are supplied• Direct measurement of transcription
factor/DNA interactions or histone
modifications• Compatible with genome-wide profiling or selective PCR-based screening• No need to optimize your own
reagents and protocol
More specific promoter analysis usingtranscription factor antibodies
ChIP has typically been performed using
antibodies directed against abundant
chromatin components, such as acetylated
histones. While this method can indicate the transcriptional state of a given
promoter, it cannot be used to identify
which transcription factor(s) were boundto the promoter(s) of interest. To do that,
ChIP must be performed using antibodies directed against specific transcription
factors. However, this is more technicallychallenging because the amount of boundtranscription factor is far less than that ofacetylated histones. To achieve success,every aspect of the ChIP procedure must be
efficient. Because the reagents included in
Active Motif’s ChIP-IT have been optimized
to work well together, you can be confidentof success, even when performing suchdifficult assays.
Controls to validate your results
ChIP is a complicated procedure that takes
several days to complete. Troubleshooting
the assay and interpreting its results canbe difficult unless you have reagents andcontrols that have proven to work in ChIP.
To help minimize your time and effort,
the ChIP-IT Kit includes a comprehensive
manual and positive and negative control
antibodies and PCR primer sets. These areideal for verifying that your DNA was fixedand sheared correctly, as well validatingthat your own antibodies and primer setsfunction in ChIP. No other kit provides sucha complete solution for ChIP. For more successful ChIP, try the ChIP-IT Kit.

Product

FormatCatalog No.

ChIP-IT™ Kit

25 rxns

53001

Toll Free — 1 877 222 9543

SEPTEMBER 2004 • VOLUME 5 • NUMBER 3

Gene Silencing

Improved Gene Silencing Through Higher Specificity Binding
Active Motif’s custom gripNA™ oligonucleotides bind to nucleic acids with higher
affinity and more stringent mismatch
discrimination than other gene silencing
reagents. This improves the results of yourgene silencing experiments by minimizing
non-specific interactions that can cause
unintended, mutant phenotypes. gripNAs
have proven to be effective at silencing genes in mammalian cells1, zebrafish,
Xenopus and in in vitro translation systems.
Because gripNAs are resistant to nucleasedegradation, they are stable in the in vivo
environment, enabling them to silence
genes expressed over a range of developmental stages. And, you can deliver
gripNAs into cells using Chariot™ II, lipids,
electroporation, microinjection and thescrape method, making gripNAs the mostversatile tool available for gene silencing.
Enhanced PNA chemistry
gripNAs are a negatively charged form ofPeptide Nucleic Acids (PNAs). PNAs are
DNA analogs in which the nucleoside bases
are attached to a synthetic backbone
rather than to deoxyribose, as in DNA. Like
DNA and RNA, PNAs bind complementary
nucleic acids by conventional Watson-Crickbase pairing. However, they hybridize totheir target with a greater affinity andan increased sequence specificity. Theirbinding affinity is significantly reduced (orcompletely eliminated) if there is evena single base-pair mismatch with theirtarget nucleic acid (Table 1). This improves
your gene silencing results by minimizing
the non-specific silencing of genes withsequences that are nearly identical, but nothomologous, to your target sequence.
Why choose gripNAs?
• Unsurpassed sequence specificity• Resistant to nucleases• Flexible synthesis modifications• Multiple delivery options• Simple online orderingwww.activemotif.com

DNA/DNATm (°C)
ΔTm (°C)

gripNA/DNA
Tm (°C)
ΔTm (°C)

No mismatches

63.9

–

58.6

–

5´-CAC-TGA-CTT-GAG-ACC-A-3´

Mismatch A

57.0

6.9

41.3

17.3

5´-CAC-TGA-GTT-GAG-ACC-A-3´

Mismatch B

49.7

14.2

No Tm

–

5´-CAC-TGA-GTG-GAG-ACC-A-3´

Sequence of DNA oligonucleotide

Mismatch C

61.8

3.1

54.6

4.0

5´-CAC-TGA-CTT-GAG-ACG-A-3´

Mismatch D

56.7

7.2

No Tm

–

5´-CGG-TGA-CTT-GAG-ACC-A-3´

Mismatch E

53.6

10.3

No Tm

–

5´-CAC-TGA-CGT-GAG-ACC-A-3´

Mismatch F

56.2

7.1

No Tm

–

5´-CAC-TGA-CTG-GAG-ACC-A-3´

Mismatch G

54.2

9.7

42.4

16.2

5´-CAC-TGA-CAT-GAG-ACC-A-3´

Table 1: Increased binding specificity of gripNAs.
Identical 16-mer gripNA and DNA probes were synthesized with the sequence 5´-TGG-TCT-CAA-GTC-AGT-G-3´. These were annealed to a complementary DNA
oligo (5´-CAC-TGA-CTT-GAG-ACC-A-3´) in Hybridization Buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 10 mM MgCl2). Each sample was heated to 90°C for 3 minutes,
then cooled gradually to room temperature. The samples were then heated at a rate of 1°C per minute from 20°C to 100°C using a thermal control unit linked to aspectrophotometer. Changes in A260 were recorded and a melting temperature (Tm) was calculated for the DNA/DNA and gripNA/DNA duplexes. The experiment
was then repeated by hybridizing the gripNA and DNA probes to a series of DNA oligos containing one- or two-base mismatches (shown above, with the mismatches underlined). Tm values were measured for each DNA/DNA and gripNA/DNA duplex and a ΔTm was calculated by subtracting the difference in the melting
temperatures of the complementary and mismatched probes. Samples that are unable to form a stable duplex generate a “No Tm” value in this assay and no ΔTm.

Hit your target only
A recurring problem of many gene silencing
reagents is a lack of specificity. This cancause unintended phenotypes because
these reagents may bind and silence
expression of more than just the intended
target. In vivo mismatch discrimination
experiments have shown that gripNA binding is reduced or completely eliminated if
there is even a single base-pair mismatch.
Experiments performed in zebrafish
embryos demonstrate how this increased
specificity can improve your results. Whilewild-type gripNA and morpholino probes
were shown to be comparable at silencing
the chordin, uroD and no tail genes, theintentional inclusion of 2 and 4 base-pairmismatches in the morpholino probes
caused non-specific effects that were notcaused by comparable gripNA probes.2

Order anytime
Active Motif’s custom gripNA synthesis
service can provide you with better materials for your gene silencing experiments. All
the information needed to design, orderand use gripNA probes can be accessed atwww.activemotif.com/gripna. Our simple,
online ordering system makes it easy toplace your gripNA order 24 hours a day, 7days a week. gripNA probes can be orderedwith or without Chariot II. In addition, you
can add a 3´ modification (Biotin, FITC or
primary amine) to your probe. Each gripNA
is verified by mass spectrophotometry andsupplied with a fluorescently labeled positive control for human CREB. To see how
increased specificity will improve your genesilencing results, try custom gripNAs today.
1. Morris et al. (2004) Gene Therapy 11:757-764.
2. Urtishak et al. (2003) Developmental Dynamics 228:405-413.

Product

FormatCatalog No.

Custom gripNA™ Probe

200 nmol

24001

Custom gripNA™ Probe w/Chariot™ II

200 nmolPrimary Amine Modification

24002
24004

Biotin Modification

24005

Fluorescein Modification

24006

Chariot™ II

96 rxns

24008

gripNA™ Human CREB Positive Control

25 nmol

24007

gripNA™ Chordin Positive Control

5 nmol24009

5

Cell-based ELISAs

continued from page 1 — Phospho-specific ELISAs for Bad, c-Src, FAK, FKHR, GSK3β, JAK & PI3K
Specificity you can count on
A concern for many researchers studying
phosphorylated proteins is the lack of
specificity of phospho-specific antibodies. To be certain that you detect only theprotein you are interested in, all antibodies in FACE Kits are stringently tested forcross-reactivity using Western Blot analysis.
All phospho-specific antibodies are verified
to detect only the activated form of thetarget protein. In addition, the phosphospecific and total antibodies are usedin tandem to ensure the phospho-specific antibody doesn’t interact with otherphosphorylated proteins. This ensures that
FACE Kits are highly specific and detect
only the protein you are interested in, atthe specific phosphorylated site.
FACE advantages• Cell-based ELISA – no extracts, gels,blotting or radioactivity• Fast – requires less than 2 hours ofhands on time
• Flexible – high-throughput chemi andcolorimetric formats• 2 Antibodies – compare phosphorylated
and native protein levels in the same kit• Quantitative results – ELISAs provide
more meaningful data than Westerns
Order today
FACE Kits provide you with a simple, efficient, quantitative method to monitor
proteins activated by phosphorylation.
No other phospho-specific assay available
offers you such a quick and convenientsolution. And, because the assay is so
sensitive, FACE Kits can accurately monitor even the smallest changes in protein
phosphorylation. Active Motif has recently
released new kits for studying the activation of Bad, c-Src, FAK, FKHR, GSK3β, JAK1
and PI3 Kinase p85, with more on the way.
To see the latest additions to this growing
product line, be sure to check our website
at www.activemotif.com/face. For the mostaccurate and convenient phospho-specificassay available, try a FACE Kit today.

6

Add anti-IgG
HRP conjugate

Add primary
antibody
3 hrs.

1 hr.

Cells arefixed following
stimulation

P

P

P

Add developing
and stop solution

Stimulatecells
Cells cultured in
96-well plate
Figure 2: Flowchart of the FACE process.
Cells are grown, stimulated and fixed in the same 96-well plate. Addition of primary and secondary antibodies detects phosphorylated protein.

Product

FormatColorimetric Kit
Catalog No.

Chemiluminescent Kit
Catalog No.

FACE™ AKT

1 x 96 rxns
5 x 96 rxns

48120
48620

48220
48720

FACE™ ATF-2

1 x 96 rxns
5 x 96 rxns

48115
48615

48215
48715

FACE™ Bad

1 x 96 rxns
5 x 96 rxns

48165
48665

48265
48765

FACE™ c-Jun (S63)

1 x 96 rxns
5 x 96 rxns

48125
48625

48225
48725

FACE™ c-Jun (S73)

1 x 96 rxns
5 x 96 rxns

48135
48635

48235
48735

FACE™ c-Src

1 x 96 rxns
5 x 96 rxns

48155
48655

48255
48755

FACE™ EGFR (Y992)

1 x 96 rxns
5 x 96 rxns

48150
48650

48250
48750

FACE™ EGFR (Y1173)

1 x 96 rxns
5 x 96 rxns

48190
48690

48290
48790

FACE™ ErbB-2 (Y877)

1 x 96 rxns
5 x 96 rxns

48130
48630

48230
48730

FACE™ ErbB-2 (Y1248)

1 x 96 rxns
5 x 96 rxns

48105
48605

48205
48705

FACE™ ERK1/2

1 x 96 rxns
5 x 96 rxns

48140
48640

48240
48740

FACE™ FAK

1 x 96 rxns
5 x 96 rxns

48145
48645

48245
48745

FACE™ FKHR (FOXO1)

1 x 96 rxns
5 x 96 rxns

48160
48660

48260
48760

FACE™ GSK3β

1 x 96 rxns
5 x 96 rxns

48170
48670

48270
48770

FACE™ MEK1/2

1 x 96 rxns
5 x 96 rxns

48180
48680

48280
48780

FACE™ JAK1

1 x 96 rxns
5 x 96 rxns

48185
48685

48285
48785

FACE™ JNK

1 x 96 rxns
5 x 96 rxns

48110
48610

48210
48710

FACE™ p38

1 x 96 rxns
5 x 96 rxns

48100
48600

48200
48700

FACE™ PI3 Kinase p85

1 x 96 rxns
5 x 96 rxns

48175
48675

48275
48775

Toll Free — 1 877 222 9543

SEPTEMBER 2004 • VOLUME 5 • NUMBER 3

Nitric Oxide Quantitation

Faster, More Accurate Measurement of Nitric Oxide Production
Active Motif’s Nitric Oxide Quantitation Kit
is a faster, more sensitive method for measuring the production of nitric oxide (NO)in your samples. The kit employs an innovative cofactor technology that reduces
the time and number of steps needed tomeasure NO levels. Plus, the Nitric Oxide
Quantitation Kit has a wider dynamic rangeof NO measurement than other methods,
which makes your results more accurate.
And, the kit can be used with a large varietyof sample types, including plasma, serum,
saliva, urine, cell lysate, tissue homogenate
and tissue culture medium.

provides an indirect measure of the original
NO concentration (Figure 1). However, thesensitivity of the Two-Step assay is limited
because NADPH, which is an essential
cofactor in step one, interferes with theGriess Reagent in step two. Assay sensitivity can be increased by the addition of athird step, in which lactate dehydrogenase(LDH) is used to eliminate excess NADPH
prior to the Griess reaction. However,while inclusion of this additional LDH stepincreases assay sensitivity, it also increases
the time required to perform each assay.

Improved accuracy for better results
In addition to its increased speed and user
friendliness, the Nitric Oxide Quantitation
Kit provides more accurate measurement
over a wider range of sample concentration than possible with other currently
used methods. The improved linearity overa broader dynamic range (Figure 2) meansthat your results will be more precise withless optimization and repetition than ispossible with any other kit.
1.2
1.0

The old way
The best index of total NO productionis the sum of both the nitrite and nitratein the sample, commonly quantified in a
Two-Step assay. The first step is conversionof nitrate to nitrite by the use of NADH- or
NADPH-dependent nitrate reductase. The
nitrite is then converted into a purplecolored azo compound by the additionof Griess Reagent. Quantitation of thisazo compound by spectrophotometry

www.activemotif.com

Nitric Oxide

Nitrate Reductase
NitriteAssay Using
Griess reagent
Figure 1: Measurement of nitric oxide by Griess Reagent.
Nitric oxide is converted to nitrite, then assayed using Griess Reagent.

Save time and money
Active Motif’s Nitric Oxide Quantitation Kit
offers a faster, easier-to-use alternative toconventional Two- and Three-Step NO assays. The kit contains a unique formulationof cofactors that accelerate the conversionof nitrate to nitrite, while simultaneously
degrading NADPH. The result is that thetime required for the reductase step isdecreased to only 30 minutes and there’s
no need for a time-consuming third step,saving you both time and effort.

OD540 nmNitrateNitric oxide is a key signaling molecule that,either directly or indirectly, stimulates hostdefenses in the immune system, maintains
blood pressure in the cardiovascular system
and modulates neural transmission in thebrain. Consequently, NO has become thesubject of extensive research. However,
because NO has an extremely short half-life(< 10 seconds), it is difficult to detect andstudy. Therefore, because NO is rapidly metabolized to nitrite and nitrate, quantitationof these stable anions is frequently used
to indirectly measure the amount of NO
originally present.

Active Motif's NO Kit
LDH method

0.8
0.6
0.4
0.2
0.0

0 5 10 15 20 25 30 35 40 45 50 55 60

Concentration of Nitrate (µM)
Figure 2: Dynamic range of nitrate standard curves.
Nitrate standard curves produced using the Nitric Oxide Quantitation
Kit and a conventional Three-Step LDH-method kit.

Get better results today
The Nitric Oxide Quantitation Kit makes
NO measurement faster, more sensitiveand easier to perform than other, moreconventional methods. In addition, its
96-well format enables straightforward
high-throughput automation. For a bettermethod of measuring NO production, try
the Nitric Oxide Quantitation Kit today!

Product

FormatCatalog No.

Nitric Oxide Quantitation Kit

2 x 96 rxns

40020

7

Competent E. coli

Efficient, Affordable Competent E. coli
RapidTrans advantages• Freedom to use anywhere from 1 to 96
reactions eliminates wasted reactions• Choice of > 1 x 108 or > 3 x 108 cfu/µg• Economical pricing (Figure 1)• Adaptable for high-throughput use

Strain & competency availability
RapidTrans TAM1 competent E. coli can bepurchased with or without the F´ and λ pir+genotype modifications depending on yourneeds. For applications that require higher
efficiencies, some RapidTrans strains areavailable in an Extra Competent format that
supplies an efficiency of > 3 x 108 cfu/µg.

Product

FormatCatalog No.

RapidTrans™ TAM1-F´ Competent E. coli

1 x 96 rxns
5 x 96 rxns

10096
10596

RapidTrans™ TAM1-F´ Extra Competent E. coli

1 x 96 rxns
5 x 96 rxns

10099
10599

RapidTrans™ TAM1 Competent E. coli

1 x 96 rxns
5 x 96 rxns

11096
11596

RapidTrans™ TAM1 Extra Competent E. coli

1 x 96 rxns
5 x 96 rxns

11099
11599

RapidTrans™ TAM1 λ pir+ Competent E. coli

1 x 96 rxns
5 x 96 rxns

11097
11597

8

$6.00

$2.00
$0.00

$3.18

$4.00

$5.57

Cost per reaction

$8.00

$3.19

Additional information
To learn more about RapidTrans CompetentE. coli, including specific genotype information, please give us a call, return the enclosed reply card or log on to our website
at www.activemotif.com/rapidtrans.

$10.00$9.90

RapidTrans™ are high-efficiency competentE. coli supplied in a convenient, 96-tube
tray. Ideal for cloning, plasmid preparationand library construction, RapidTrans cells
are affordably priced and packaged in aformat that provides maximum flexibility
while eliminating waste. Each tube contains
50 µl of cells for one transformation reaction. In contrast to other 96-well formats
that require thawing all 96 reactions at one
time, RapidTrans enables the use of as few
or as many reactions as needed, without
thawing the other cells. This eliminates the
reduced efficiencies and waste caused byrepeated freeze/thaw cycles.

Company I Company S* Company N** Active Motif* You must purchase 4 plates at a time.
** Only 20 µl of cells/rxn are supplied.

Figure 1: RapidTrans Competent E. coli are priced economically.
The graph above shows the cost per reaction of 96-tube competentE. coli from various manufacturers.

Toll Free — 1 877 222 9543

CONTACT INFO
U.S.
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
Email: tech_service@activemotif.com

EUROPE
104 Avenue Franklin-Box 25
B-1330 Rixensart, Belgium
Toll Free: +800 222 95 430
Direct: +32 (0)2 653 0001
Fax: +32 (0)2 653 0050
Email: eurotech@activemotif.com

PRSRT STD
US POSTAGEPAID
PERMIT 236
92054