THE NEWSLETTER OF ACTIVE MOTIF — May 2006 • volume 7 • number 2

AlmaKnowledgeServer
Uncovers Hidden
Knowledge from
Medline2 NEW: Speciﬁc, Flexible Labeling of Proteins

IN
THIS
ISSUE

4 Detect Protein Phosphorylation Directly Inside the Cell
5 NEW: Highly Speciﬁc Kinase Activity Assays
6 Application Note: Quantitation of ActivatedTranscription Factors
7 ChIP-IT™ Simpliﬁes Chromatin Immunoprecipitation
8 MethylDetector™ – Fast & Efﬁcient DNA Methylation
Analysis

8 Co-Immunoprecipitation of Nuclear Protein
Complexes
9 Deliver Functional Proteins Directly into Living Cells
10 NEW: Effective Fluorescent Labels for Bioanalysis
11 Tap into the Knowledge Stored in Medline with
AlmaKnowledgeServer
12 Optimized Preparation of Cellular Samples

Protein Labeling & Translocation

NEW: Speciﬁc, Flexible Labeling of Proteins
Active Motif’s new LigandLink™* Universal Labeling technology is an innovative tool for performing site-speciﬁc labeling of proteins in living cells. With
LigandLink, it is now possible to create a single fusion protein that can belabeled with a variety of tags, making it ideal for use in FRET and co-localization studies, where the requirement for varying ﬂuorophores is high.

The LigandLink method
In the LigandLink method, the geneof interest is cloned in frame with thegene for E. coli dihydrofolate reductase(eDHFR) in the pLL-1 vector (Figure 1).
The vector is then transfected into

Prokaryotic protein for a cleaner signal
The LigandLink protein is derived fromthe E. coli DHFR protein and binds rapidpCM
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pLL-1

SV 4

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eDHFR

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HSV TK poly A

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However, protein function is commonly
regulated via protein-protein interactions, creating the need for multiple
labeling systems. Using conventional
approaches, this means you would needto clone a series of constructs containing your gene of interest fused todifferent ﬂuorescent proteins. This has
made such studies time-consuming andlabor intensive. In contrast, LigandLink
is a novel tool that makes it possible tocreate a single clone that can be labeledin cells with different tags. This makes
LigandLink an ideal tool for co-localization studies, where multiple labelingcolors are required.

User-friendly vector
The LigandLink vector, pLL-1 (Figure 1), iseasy to use. It has a CMV promoter toensure high-level expression of eDHFR
fusion proteins, with Neomycin for
selection of stable cell lines. In addition, the multiple cloning site (MCS) was
designed to facilitate cloning, whatever
method you use. In addition to many
popular restriction sites, the MCS
includes three blunt-cutting restriction
enzymes towards the 3´ end, each in adifferent reading frame with the eDHFR
gene. This enables a number of PCR and
restriction enzyme cloning strategies.

Age I“ LigandLink’s ease of use makes it
an ideal tool for co-localization
experiments where multiple
labeling colors are required.”Why use LigandLink?
• Spend less time on cloning andclone validation – clone only once,but obtain different functionalities
by using different LigandLink labels• Superior ﬂuorescence – syntheticdyes have improved spectral properties vs. naturally ﬂuorescent proteins• Small label – unlikely to interferewith protein function• Label your protein in living cells –LigandLink labels are cell permeable

RI
Eco

Improved co-localization studies
The ability to label proteins in living cells
is key to understanding the dynamics
and functions of proteins. Classically,
these studies have been performed byfusing a gene of interest to a ﬂuorescentprotein, such as green ﬂuorescent protein (GFP), and transfecting the construct
into cells. These approaches have signiﬁcantly expanded our understanding ofprotein function within the cell.

mammalian cells and used to express
the fusion protein. Twenty-four hours
post-transfection, the protein of interest
can be labeled simply by adding theLigandLink ligand of choice to the cell
medium (Figure 2). Depending on thecell type and the label used, cells can beimaged in as little as 10 minutes.

eo
S V 4 0 o ri

P

Figure 1: The LigandLink pLL-1 vector.

2

Toll Free — 1 877 222 9543

May 2006 • volume 7 • number 2

Protein Labeling & Translocationeasier, we have already prepared a line ofpre-made vectors containing transcription factors such as NFκB, STAT and
p53 that are ready to transfect into themammalian cell line of your choice.

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Figure 2: Speciﬁc protein labeling using LigandLink.
The gene of interest is cloned into pLL-1 in frame with the vector’s E. coli dihydrofolate reductase (eDHFR) gene. Aftertransfection into cells, the protein of interest is expressed as a fusion to eDHFR. Addition of cell-permeable LigandLink Labelto the medium results in rapid, specific binding of the label by the fusion protein.

ly and speciﬁcally to the LigandLink TMP(trimethoprim) ligand. This is because
TMP is an antibiotic that was designed
to speciﬁcally inhibit the bacterial
eDHFR protein, without interacting withmammalian proteins. The result is highspeciﬁcity binding (Kl ≈ 1 nm) with minimal background (Figure 3). In addition,
the eDHFR fusion protein is signiﬁcantly
smaller than naturally ﬂuorescent proteins, such as GFP (18 kDa vs. 27 kDa for
GFP), which means that cloned protein
activity is less likely to be affected.
Superior and ﬂexible dye options
Once a gene of interest is cloned into aclassical ﬂuorescent protein-fusion vector, it can be time consuming and laborious to change the ﬂuorescent fusion.

Figure 3: Membrane-localized eDHFR labeling by
LigandLink Hexachloroﬂuorescein.
The myristoylation/palmitoylation sequence from Lyn
was cloned into pLL-1 and transfected into CHO cells.
Twenty-four hours post-transfection, 10 nM LigandLink
Hexachlorofluorescein was added to the cells; 2 hours
later, the above image was taken.

www.activemotif.com

In addition, native ﬂuorescent proteins
are limited by their intrinsic physicalproperties and may suffer from highlevels of photobleaching, low quantum
yield or a number of other physicallimitations. The dyes used in LigandLink
have been speciﬁcally engineered topossess superior physical properties thatare ideal for use in common applications
such as FRET and co-localization research(Figure 4).
Pre-made translocation vectors
The LigandLink Universal Labeling technology is ideal for studying translocation
events because of the highly permeable
nature of the TMP ligand and the simple
way in which proteins are labeled inin vivo environments. To make it evenFigure 4: Translocation of NFκB p65 to the nucleus.
The LigandLink pLL-1-NFκB p65 Kit was used to express
and monitor the translocation of NFκB into the
nucleus. HeLa cells were stimulated with TPA + CI andimages were captured 2 hours after stimulation.

Try LigandLink today
LigandLink will improve the way youlabel proteins in living cells. The highly
speciﬁc and stable LigandLink tags are
ideal for use in FRET and co-localization
research because once a protein of interest is expressed as a LigandLink fusion,
you can easily change the properties of
its tag by adding a different LigandLinklabel to the medium. Find out more
about the new LigandLink system byvisiting www.activemotif.com.

Product

FormatCatalog No.

LigandLink™ pLL-1 Kit

1 kit

34001

LigandLink™ pLL-1-AKT1 Kit

1 kit

34002

LigandLink™ pLL-1-AKT2 Kit

1 kit

34003

LigandLink™ pLL-1-NFκB p65 Kit

1 kit

34004

LigandLink™ pLL-1-p53 Kit

1 kit

34005

LigandLink™ pLL-1-STAT1 Kit

1 kit

34006

LigandLink™ Fluorescein Label

300 rxns

34101

LigandLink™ Hexachloroﬂuorescein Label

300 rxns

34104* Patent pending.

3

Protein Phosphorylation

Detect Protein Phosphorylation Directly Inside the Cell

The FACE method – an “in-cell” Western
In FACE, cells are cultured as desired in 96well plates and stimulated to induce thepathway of interest. Following stimulation,
the cells are rapidly ﬁxed, which preserves
activation-speciﬁc protein modiﬁcations.
Each well is then incubated with a primary
antibody speciﬁc for the protein modiﬁcation of interest. Subsequent incubation
with secondary HRP-conjugated antibody
and developing solution provides a colorimetric or chemiluminescent readout thatis quantitative and reproducible (Figure 1).

No more cell extractions
Because FACE Kits are cell-based, you
no longer need to prepare cell extracts
and perform Westerns to determine thephosphorylation state of your protein.
Plus, as FACE Kits enable you to ﬁx yourcells directly after stimulation, further
protein modiﬁcations that can occur
during the extraction process are eliminated. This means you get more accurate
results, in a fraction of the time, as FACE
Kits require less than 2 hours of hands ontime to perform.
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
Flow chart 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.

FACE™ Product Line
FACE™ AKT (S473)

FACE™ ATF-2 (T71)

FACE™ Bad (S112)

FACE™ c-Jun (S63)

FACE™ c-Jun (S73)

FACE™ c-Src (Y418)

FACE™ EGFR (Y992)

FACE™ EGFR (Y1173)

FACE™ ErbB-2 (Y877)

FACE™ ErbB-2 (Y1248)

FACE™ ERK1/2 (T202/
Y204 & T185/Y187)

FACE™ FAK (Y397)

FACE™ FKHR (T24)

FACE™ GSK3β (S9)

FACE™ JAK1 (Y1022/
Y1023)

FACE™ JNK (T183/Y185)

FACE™ MEK1/2 (S217/
S221)

FACE™ NFκB Proﬁler(S468 & S536)

FACE™ p38 (T180/Y182)

FACE™ PI3 Kinase p85

FACE™ STAT2 (Y689)

FACE™ STAT4 (Y693)

FACE™ STAT6 (Y641)

4

0.6

Phospho JNK
Total JNK

0.5

OD450 nmFast Activated Cell-based ELISA (FACE™) Kits provide you with a simple, sensitive, cell-based method for monitoring protein phosphorylation directly in thecell without the need for cell extracts, gels or membrane blotting.

0.4
0.3
0.2
0.1

0

0

0.1
10
1
Anisomycin induction (µg/ml)

Figure 1: Monitoring the effects of anisomycin treatment
on JNK phosphorylation.
The FACE JNK assay was used to assay the levels ofphosphorylated JNK in response to different concentrations of anisomycin treatment on macrophage 4/4 cells.

FACE advantages• Cell-based method – no extracts,
gels or membrane blotting• Fast – requires less than 2 hours ofhands on time
• Flexible – use suspension or
adherent cells• Total & Phospho Antibodies –compare both phosphorylated andnative protein levels in the same kit• Quantitative results“FACE Kits enable detection ofphosphorylated proteins within thecell, eliminating the need for cell
extractions, gels & Western blotting.”
A variety of kits to choose from
FACE Kits are available for many kinases
and receptors, as indicated in the table
at left. All kits are conveniently provided
in both colorimetric and ultra-sensitive
chemiluminescent formats. For more
information about the FACE Kits, please
visit www.activemotif.com/face.

Toll Free — 1 877 222 9543

May 2006 • volume 7 • number 2

Kinase Assays

NEW: Highly Speciﬁc Kinase Activity Assays
Active Motif’s KineActive™ activity assays enable the study of highly conserved members of kinase families in cell and tissue extracts by providing
you with unmatched sensitivity and speciﬁcity in a quantitative, non-radioactive, high-throughput format.
Improved technique
Traditionally, kinase activity has beenstudied through the use of radioactive
immunoprecipitation-based kinase assaysthat can be time-consuming and lacksensitivity. In contrast, KineActive Kits
make it simple to assay kinase activity
in less than 5 hours in a non-radioactive,
96-stripwell format (See Flow Chart).
Plus, because KineActive Kits are very
sensitive, you can assay all sample types,
including cell lines, tissues and puriﬁedproteins. Try KineActive today for an improved method to study kinase activity.

“KineActive provides speciﬁc
detection of activated kinases in a
fast, non-radioactive format.”
Advantages• Quantitative• Highly speciﬁc (see Figure 1)• Non-radioactive detection• High-throughput stripwell format• Includes sample preparation
reagents and protein standards• Improved sensitivity• Assay cell and tissue extracts or
puriﬁed proteinwww.activemotif.com

BiotinPhosphorylated
peptidesubstrate

ATP

DesiredkinaseStreptavidin-HRP
conjugate

P

Kinaseantibody

Phospho
Peptide antibody

Flow chart of KineActive process.
In KineActive, sample containing the desired kinase isadded to a 96-well antibody capture plate. This is followed by addition of kinase antibody, which binds tothe plate and specifically captures the kinase of interest. After washing, a biotinylated, non-phosphorylated
peptide substrate is added in the presence of ATP.
The immobilized kinase phosphorylates this peptide,which is captured by the addition of a second antibody
directed against the phosphorylated peptide. Additionof Streptavidin-HRP and developing solution provides aquantitative colorimetric readout.

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Detect only the kinase you want
Many kinase families, such as Src, containmembers that phosphorylate a common target. Therefore, to accurately
measure the activity of a speciﬁc kinase,
it is necessary to immunoprecipitate itfrom others that can act upon the same
peptide substrate. The high speciﬁcityof the KineActive Kits enable the studyof conserved kinase families with ease.
To illustrate, recombinant proteins for
Src, Yes, Lyn, Fyn, Hck, Lck, Brk and FGR
were incubated in KineActive and tested
for cross-reactivity using the Src, Yes andLyn primary antibodies used in the kits.
As the data clearly illustrates, the antibodies used in KineActive are speciﬁc for
only the kinase of interest (Figure 1).

Non-phospho
peptide substrate������������
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Figure 1: Speciﬁc detection of Src family members using KineActive.
Recombinant proteins for Src, Yes, Lyn, Fyn, Hck, Lck, Brk and FGR were incubated in KineActive and tested for specificity
using the Src, Yes and Lyn primary antibodies. As the data clearly illustrates, the antibodies used in KineActive are specificfor only the factor of interest. Note: the enzymes for Fyn, Hck, Lck, Brk and FGR were tested for activity using radioactive
kinase assays in separate experiments.

Product

FormatCatalog No.

KineActive™ Lyn

1 x 96 rxns

56096

KineActive™ Src

1 x 96 rxns

56196

KineActive™ Yes

1 x 96 rxns

56296

5

Transcription Factor ELISAs

Application Note: Quantitation of Activated Transcription Factors
To date, screening large numbers of drug compounds to determine effects
on transcription factor activation has been limited by a lack of suitableassays. Existing methods such as Westerns, EMSA and reporter assays lacksensitivity & reproducibility and require substantial processing. In this study,
the TransAM™ Kit, an ELISA-based method that assays transcription factor
activation1, is used to examine translocation and DNA-binding activity oftranscription factors. Competition assays were also performed.
Materials & Methods
Detection of DNA-binding by ELISAThe TransAM™ NFκB p50 Kit was used
to assay DNA-binding activity of NFκB
p50 in nuclear and cytoplasmic extracts
made from both unstimulated and TNF
stimulated HeLa cells. For competitive
binding experiments, 5 µg of nuclear extract from stimulated cells were assayedin the presence of wild-type or mutatedcompetitor oligonucleotides (oligos).
Each TransAM Kit contains a 96-wellplate comprised of twelve 8-well strips.
Each well is supplied coated with oligothat contains a consensus-binding site
for NFκB (5´-GGGACTTTCC-3´). Thissequence is also present in the wild-typecompetitor oligo. The mutated competitor oligo contains three mutated bases,
destroying the NFκB binding site. The
p50 and secondary antibodies, as well as
Wash & Binding Buffers and Developing
Solutions were supplied in TransAM.

Figure 2: Speciﬁcity of TransAM Kits.
TransAM assays are performed in the presence of wildtype and mutated competitor oligonucleotides using 10µg/well of cellular extract.

The TransAM procedure
1. Bind transcription factor to consensus site.
30 µl of Binding Buffer was added toeach well. For competitive binding
experiments, Binding Buffer contained
1, 5 or 20 pmol of wild-type or mutatedcompetitor oligos. Increasing amounts of
HeLa nuclear and cytoplasmic extracts
were diluted in 20 µl Lysis Buffer andadded to wells being used. After a 1 hourroom temperature (RT) incubation, wells
were washed 3X with Wash Buffer.
2. Binding of primary antibody.
100 µl of a 1:1000 dilution of NFκB p50
rabbit pAb was added to each well and incubated at RT for 1 hour. After incubation,
wells were washed 3X with Wash Buffer.

Figure 1: TransAM measures DNA-binding activity.
Nuclear and cytoplasmic extracts were assayed using
the TransAM NFκB p50 Kit. Because activated NFκB
translocates to the nucleus, only nuclear extract fromstimulated cells should contain activated NFκB.

6

3. Binding of secondary antibody.
100 µl of a 1:1000 dilution of HRP-conjugatedsecondary antibody was added to eachwell and incubated at RT for 1 hour. Afterincubation, wells were washed 3X with
Wash Buffer.

4. Colorimetric reaction.
100 µl TMB substrate was added toeach well for 10 minutes before adding 100 µl Stop Solution. Optical density was then read at 450 nm using aspectrophotometer. All results wereexpressed as averages of duplicates assayed after subtracting the blank values.
Results
Activation & Translocation of NFκB
Figure 1 demonstrates TransAM measures
DNA-binding activity of NFκB. The
linearity shows that the signal is directly
proportional to the quantity of transcription factor. The assay also conﬁrms
that stimulation results in both translocation and activation of DNA-binding
activity of NFκB as only nuclear extracts
from stimulated cells test positive.
Assay Speciﬁcity
The speciﬁcity of the transcription factor/DNA interaction is conﬁrmed throughuse of the competitor oligos. Figure 2 demonstrates that increasing amounts of wildtype competitor oligo reduce the signal of
the nuclear extract. In contrast, addition ofa large excess of mutated competitor oligo
does not affect the signal strength.
Conclusion
Because TransAM speciﬁcally measurestranscription factor bound to its target
DNA, it quantiﬁes transcription factor
activation. While the extreme complexityof transcriptional regulation dictates thatmultiple study systems and numerous assay
methods be employed in drug discovery
efforts, TransAM’s unique features (highthroughput, compatible with both cells andtissues, high-sensitivity) make it a powerful
addition to existing methods for studying
transcription factor activation. TransAM
Kits are now available for 30 different factors. Visit www.activemotif.com/transam.
Reference: 1. Renard P. et al. (2001) Nucleic Acids Research 29(4): E21.

Toll Free — 1 877 222 9543

May 2006 • volume 7 • number 2

Chromatin Immunoprecipitation

ChIP-IT™ Simpliﬁes Chromatin Immunoprecipitation
Active Motif’s kits for chromatin immunoprecipitation (ChIP) simplify yourChIP experiments by providing you with ready-to-use reagents, including
shearing and reaction buffers, positive control antibodies and PCR primers,
DNA puriﬁcation columns and a comprehensive protocol to ensure success.
How ChIP works
Chromatin immunoprecipitation is a
powerful tool for analyzing genome
regulation. In ChIP-IT, intact cells are ﬁxed
using formaldehyde, which cross-links andpreserves protein/DNA interactions. The
DNA is then sheared into small, uniform
fragments and protein/DNA complexes
are immunoprecipitated using antibodies
directed against the protein(s) of interest.
Next, the cross-links are reversed and DNA
fragments are puriﬁed and screened todetermine which gene, or group of genes,
were bound by the protein of interest.

“ChIP-IT improves ChIP experiments
by providing ready-to use reagentsand proven controls in a single kit.”

ChIP-IT advantages• Easy – includes critical buffers,
Protein G and puriﬁcation columns• No need to optimize reagents andprotocol• Your choice of enzymatic orsonication shearing• Direct measurement of transcription
factor/DNA interactions or histone
modiﬁcations
Choose enzymatic or sonication shearing
A successful ChIP begins with correctly
shearing DNA into 200-1000 bp fragments.
This can be achieved using either enzymatic
digestion or sonication. Sonication is aneffective method for shearing DNA but
can be difﬁcult to optimize. In contrast,
enzymatic digestion can be used to quickly
and easily digest DNA into fragments suitable for ChIP with very little effort. For yourconvenience, ChIP-IT Kits are available witheither sonication or enzymatic shearing.
Order a ChIP-IT Kit today
Active Motif’s ChIP-IT Kits provide youwith everything you need to makeyour ChIP experiments easier and more
reproducible. For more information, visit
www.activemotif.com/chip.

1000 bp
500 bp

Save on Antibodies for ChIP
Finding antibodies that have been
proven to work in ChIP is not easy.
Although our list of ChIP-validated
antibodies is growing (see Table 1),
we need your help. That’s why inthe months of May and June we areoffering 50% off the purchase of
any antibody that is purchased inconjunction with one of our ChIP-IT
Kits. Visit www.activemotif.com tobrowse our antibody search engine.
Antibody

Cat. No.

Price ($US)

AP-2 pAb

39304

280

c-Jun pAb

39309

280

C/EBPα pAb

39306

280

DNMT1 mAb

39204

280

DNMT3A mAb

39206

280

DNMT3B mAb

39207

280

E2F-1 pAb

39313

280

E2F-6 mAb

39509

280

GATA-1 pAb

39025

280

HBP-1 mAb

39511

280

HDAC3 pAb

40968

280

HDAC4 pAb

40969

280

HDAC5 pAb

40970

280

HDAC6 pAb

40971

280

IRF-3 pAb

39033

280

JunB pAb

39326

280

JunD pAb

39328

280

p53 pAb

39334

280

Pax-5 pAb

39336

280

PPARγ pAb

39338

280

RNA pol II mAb

39097

280

Sp1 pAb

39058

280

TRF2 pAb

39223

280

Table 1: ChIP-validated antibodies available.

100 bp
1

2

3

4

5

Figure 1: Analysis of DNA sheared using the Enzymatic
Shearing Kit.
HeLa cells were fixed for 10 minutes with 1% formaldehyde and then chromatin was prepared using the
Enzymatic Shearing Kit protocol. Chromatin was shearedwith the enzymatic shearing cocktail for 5, 10 & 15 minutes and the reaction was stopped.
Lane 1: 100 to 1000 bp ladder.
Lane 2: Unsheared HeLa DNA.
Lane 3: HeLa DNA treated for 5 minutes.
Lane 4: HeLa DNA treated for 10 minutes (optimized).
Lane 5: HeLa DNA treated for 15 minutes.

www.activemotif.com

Product

FormatCatalog No.

ChIP-IT™

25 rxns

53001

ChIP-IT™ w/o controls

25 rxns

53004

ChIP-IT™ Shearing Kit (included in 53001 & 53004)

10 rxns

53002

ChIP-IT™ Enzymatic

25 rxns

53006

ChIP-IT™ Enzymatic w/o controls

25 rxns

53007

Enzymatic Shearing Kit (included in 53006 & 53007)

10 rxns

53005

7

DNA Methylation & Co-IP

MethylDetector™ – Fast & Efﬁcient DNA Methylation Analysis
Active Motif’s MethylDetector™ Bisulﬁte Modiﬁcation Kit makes DNA
methylation analysis fast and efﬁcient by combining optimized reagents for
performing DNA conversion with time-saving DNA puriﬁcation columns andpositive control PCR primers to help you validate your results.
Proven controls ensure success
DNA methylation analysis typicallyinvolves using bisulﬁte to convert
unmethylated cytosines to uracils, whileleaving methylated cytosines unchanged.
The DNA is then ampliﬁed by PCR andanalyzed by sequencing or restriction
digest. However, bisulﬁte conversion
can be technically challenging, andconﬁrming the process was successful
before sample analysis is preferred.
MethylDetector provides positive
control PCR primers that are speciﬁc
for bisulﬁte-converted DNA, so you canconﬁrm the procedure worked before
starting further costly analysis (Figure 1).

Advantages• Flexible – use high G/C content
sequences and uncut DNA• Reproducible – 99% conversion
efﬁciency• Efﬁcient – reagents and protocol
with proven controls• Easy – DNA puriﬁcation columns
mean no more precipitations
Try MethylDetector today
MethylDetector will speed and simplify
your methylation analysis. Learn more atwww.activemotif.com.

M

1

2

3

4

5

500 bp400 bp300 bp200 bp100 bpFigure 1: Agarose gel analysis of PCR products generated
with MethylDetector.
Three different DNA conversions were performed(Lanes: 1-3) and compared to an unconverted DNA
control (Lane: 5) and to a no DNA control (Lane: 4). The
presence of PCR product in only the converted samples
demonstrates the conversion efficiency and reproducibility of the MethylDetector Kit.

Product

FormatCatalog No.

MethylDetector™

50 rxns

55001

Co-Immunoprecipitation of Nuclear Protein Complexes
Performing co-immunoprecipitation of nuclear protein complexes has justgotten easier as Active Motif’s Nuclear Complex Co-IP Kit provides optimized
reagents that maintain DNA-binding protein complexes and improve results.
Optimized method and reagents
Co-immunoprecipitation (Co-IP) isoften used to analyze protein/protein
interactions. However, traditional Co-IP
methods are not optimal for studying
DNA-binding protein complexes as thesecomplexes are unstable. The Nuclear
Complex Co-IP Kit helps maintain thesecomplexes by providing optimized reagents for both nuclear extract preparation and immunoprecipitation, including
both high and low stringency buffers, aswell as additional salt and detergent tomodify stringency as required.

8

Advantages• Simple and efﬁcient• Optimized extraction procedure
preserves nuclear protein complexes• Easily alter IP stringency to detect
interactions of varying strengths
Order the new Co-IP Kit today
The Nuclear Complex Co-IP Kit enables
you to study tightly bound or weakprotein complexes with equal ease. Visit
www.activemotif.com.

250
1

2

3

Figure 1: Western blot analysis of the IP’d p33 subunitof the RNA pol II complex.
For IP experiments, the stringency of the IP High
Buffer was increased by supplementing with NaCl andDetergent. 100 µg of HeLa nuclear extract was used per
IP and incubated with either 2 µg p33 antibody or no
antibody. Following the IP, Western blot using RNA pol
II mAb was used to detect the p33/RNA pol II complex(lane 3). This demonstrates the Co-IP was successful inmaintaining the protein complex.
Lane 1: Western blot control
Lane 2: Negative Control (no antibody used in IP)
Lane 3: Co-IP: IP using p33/WB using RNA pol II

Product

FormatCatalog No.

Nuclear Complex Co-IP Kit

50 rxns

54001

Toll Free — 1 877 222 9543

May 2006 • volume 7 • number 2

Protein Transfection

Deliver Functional Proteins Directly into Living Cells
Chariot™ is Active Motif’s patented* protein delivery reagent that efﬁciently
transports biologically active proteins, peptides and antibodies directly into
cultured mammalian cells. Delivery is complete in less than two hours andprovides efﬁciencies of 65-95%. After delivery, living cells can be assayed
immediately to determine the effects of the introduced material. Thesefeatures make Chariot an ideal tool for a variety of functional studies.
Targeted delivery
Chariot is a peptide that forms a noncovalent complex when incubatedwith your puriﬁed protein, peptide
or antibody for 30 minutes at room
temperature. Addition of the complex
to cells results in its rapid internalization. Once inside the cell, the complex
dissociates and Chariot is transported to
the nucleus, while the delivered protein
is biologically active and free to proceed
to its cellular target (Figure 1).

Deliver biologically active proteins
The ability of Chariot to deliver biologically active protein is shown using
a 119 kDa subunit of β-galactosidase.
β-galactosidase is composed of four
subunits that must assemble to form
functional protein. HeLa cells turn blue
when X-gal is added after delivery of theChariot-galactosidase complex, demonstrating successful delivery of functional
β-galactosidase (Figure 2).

Advantages• Delivers active protein directly into
living cells• Up to 95% efﬁciency in < 2 hours• Works in a variety of cell lines, aswell as in vivo
• Study living cells – no ﬁxing needed
Why use protein delivery?
Direct delivery of active protein makes
it easy to perform studies not evenpossible using DNA transfection andexpression. Successful Chariot deliveryof proteins, peptides and antibodies has
been shown in a wide range of cell lines,
including hard-to-transfect neuronal, primary and plant cells. For a list of papers
that cite the use of Chariot, simply returnthe enclosed reply card or download thelist at www.activemotif.com/chariot.

“The ability to deliver active proteins directly into living cells and then assay
their effect makes feasible studies not even possible with other methods.”
Chariot delivers results
Chariot speeds and simpliﬁes a variety offunctional studies because it efﬁciently delivers biologically active proteins, peptides
and antibodies directly into mammaliancells, even into hard-to-transfect and nondividing cells. To learn what your protein isreally doing, study it using Chariot.

Figure 1: Targeted protein delivery.
50 ng of a 10 kDa nuclear protein that was labeled with
Lucifer yellow at the C-terminus was complexed with
Chariot and delivered into HS-68 cells. Unfixed cells
were observed 90 minutes post-delivery.

Non-covalent delivery of native protein
Many protein delivery systems requirethat you begin by fusing a carrier protein
to your macromolecule. In addition to
being time-consuming, this can alter thefolding characteristics of your protein
and, ultimately, its function. Because
Chariot forms a non-covalent bond withyour protein, it does not affect the delivered protein’s folding or function.

www.activemotif.com

Figure 2: Chariot delivery of β-galactosidase.
One µg of a 119 kDa subunit of β-galactosidase wascomplexed with Chariot for 30 minutes and delivered
into HeLa cells. Cells were fixed and stained with X-gal
2 hours post-delivery.

Product

FormatCatalog No.

Chariot™

25 rxns**
100 rxns

30025
30100

β-Galactosidase Staining Kit

75 rxns

35001* Chariot is covered under U.S. Patent No. 6,841,535. Purchase includes the right to use for basic
research purposes only. Other-use licenses are available; please contact Active Motif Technical
Services for additional information.
** A rxn is deﬁned as sufﬁcient Chariot reagent to deliver protein to cells in a 35 mm plate.

9Fluorescent Detection

NEW: Effective Fluorescent Labels for Bioanalysis
Active Motif’s Chromeo™ Dyes improve labeling of biomolecules for cell
culture experiments because they exhibit excellent photostability, pH tolerance, low cell toxicity and can be used with most excitation sources.
Stokes shift of 124 nm, making it an ideal
partner for multiplexing with other, shortshifted 488-excitable dyes.
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Recent advances in ﬂuorescent chemistry and ﬂuorescent detection equipment have facilitated a rapid increase in
the development of synthetic dyes foranalysis of biomolecules. Fluorescentdyes are widely used for cell identiﬁcation, ﬂow cytometry and cellular imagingof localization and movement of subcellular structures. For a ﬂuorescent dye tobe useful in ﬂuorescence microscopy it
must possess many key features: brightness of conjugates, photostability, solubility and pH insensitivity. In addition,
detection limits that are signiﬁcantly
improved compared with colorimetricbased detection platforms is essential.

Chromeo™ Dye advantages• Convenient spectral properties• High quantum yields• pH insensitive• Available as reactive NHS-Esters to label
amino groups or as carboxylic acid
• Unmatched photostability����������
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Visit the Active Motif Chromeon website
As a leader in providing ﬂuorescent tools
for bioanalysis, Active Motif Chromeon
also offers several other dyes to meetyour research needs, including RuLabels
and Py-Dyes. RuLabels are phosphorescent ruthenium probes that emit strong
polarized light with a long decay time,
which enhances sensitivity and reduces
background. The Py-Dyes possess unique
physical and spectral properties thatmake conjugation simple, while greatly
reducing background. To ﬁnd out more
about Active Motif Chromeon products
visit www.chromeon.com.

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Figure 2: Photostability test.
Dyes were dissolved in PBS (22 mM, pH 7.2) and thesolutions were allowed to stand at room temperature
in daylight. Absorbance at the respective maximum wasmeasured over a period of 36 days.

Absorption

Emissionε L/(molcm)

Quantum
Yield (%)*

Stokes
Shift

Chromeo™ 494

494

628

20,00025

124 nmChromeo™ 546

545

561

96,8001016 nm

Chromeo™ 642

642

660

180,000

15

18 nm

Dye

Table 1: Chromeo Dye properties. (* when conjugated to BSA)

Figure 1: Cytosolic staining of HeLa cells using
Chromeo 494.

Excellent ﬂuorescent properties
The Chromeo Dyes, Chromeo 494,
Chromeo 546 and Chromeo 642, exhibit
many key features that make them ideal
for any ﬂuorescent application including
superior luminescence properties andbroad Stokes shifts (Table 1). Chromeo
Dyes are also suitable for use with many
common excitation sources such as diodelasers, LEDs, tungsten and Xenon arc lamps.
In addition, Chromeo 494 has a very large

10

Product

FormatCatalog No.

Chromeo™ 494 Carboxylic Acid

1 mg
5 mg

15110
16110

Chromeo™ 494 NHS-Ester

1 mg
5 mg

15111
16111

Chromeo™ 546 Carboxylic Acid

1 mg
5 mg

15210
16210

Chromeo™ 546 NHS-Ester

1 mg
5 mg

15211
16211

Chromeo™ 642 Carboxylic Acid

1 mg
5 mg

15310
16310

Chromeo™ 642 NHS-Ester

1 mg
5 mg

15311
16311

You can ﬁnd additional Chromeon Dyes at www.chromeon.com

Toll Free — 1 877 222 9543

May 2006 • volume 7 • number 2

Knowledge Mining

Tap into the Knowledge Stored in Medline with AlmaKnowledgeServer
AlmaKnowledgeServer (AKS) is a powerful text mining system that makes it
possible for you to uncover relationships that exist in the scientiﬁc literature
between genes, proteins, chemical compounds and diseases. AKS can evendiscover links that exist, but that have not yet been published.
Keep current with the latest papers
Researchers read journal articles to learn
about new developments in their ﬁeld.
But, with 1000’s of journals and morethan 15 million abstracts on Medline(plus over 2,000 more added daily), it isphysically impossible to keep current,
let alone to explore all of the areas thatmay provide new and useful insights.
The answer is AlmaKnowledgeServer(AKS), a powerful text mining systemthat, unlike you, reads every paper everyday. AKS examines all abstracts added
to PubMed, then applies statistics andrules-based analysis to uncover relationships that exist in the scientiﬁc literature
between genes, proteins, chemical compounds and diseases. This enables you tomake better informed decisions about
your research in far less time.
Bioentity and synonym recognition
AKS understands and retrieves biologicalconcepts (bioentities) instead of simple
words. The bioentities recognized by
AKS include genes and proteins, diseases
and chemical compounds. AKS has beentaught the many synonyms, acronyms
and other scientiﬁc naming ambiguities
that plague traditional searching. For
example, searching PubMed for IL-2, IL2,
IL 2 or interleukin 2 return different
results, though all are the same gene.
So, an exhaustive search for a single
gene may actually require 5-10 individual
searches. With AKS, one search returns
all documents that are relevant to IL-2.

Learn by exploring co-occurrences
After a search, AKS ranks the relevant
concepts and bioentities. Documents
can be explored by viewing sentencesin which two or more bioentities occur(Figure 1). This makes it possible to build
a signiﬁcant understanding of the interrelations between bioentities before
you’ve even read any of the papers. You
can also read abstracts with co-occurring bioentities highlighted, and apply
ﬁlters to ﬁne tune your results. And, AKS
provides links to Medline, so you canquickly access the papers of interest.

What’s a picture worth? 1000’s of papers
Because knowledge often comes by
seeing data from a different perspective, AKS also makes it possible to view
bioentity relationships graphically. Figure
2 diagrams the relationships between
co-occurring bioentities. The power ofthe system is that even tough this visual
representation contains the content of
thousands of documents, the relationships are easily seen, helping you to
obtain new insights with minimal effort.
Information for better decision making
AKS can help provide answers that areimportant to your research, like which
proteins are involved in a disease, or who
are the thought leaders in an emerging
ﬁeld? To learn more, give us a call or visit
Active Motif’s biocomputing division,
TimeLogic, at www.timelogic.com.

Figure 1: Sentences displaying co-occurrences of “leptin” and “obesity” bioentities.

Figure 2: AKS includes a powerful visual environment to map relationships between bioentities.

www.activemotif.com

11

Sample Preparation

Optimized Preparation of Cellular Samples
Active Motif provides many kits that are ideal for preparing and quantifying
samples from cell or tissues samples. These include our popular Nuclear Extract Kit,
Mitochondrial Fractionation Kit, GAPDH Whole-cell Normalization Kit and ProStain™
Protein Quantiﬁcation Kit.
Nuclear Extract Kit
The Nuclear Extract Kit is ideal for preparing nuclear, cytoplasmic and whole-cell
extracts from mammalian cells and tissues
because it eliminates the need to optimize
reagents while ensuring consistently highyields. The resultant high-quality extracts
may be used with Active Motif’s TransAM™
Kits, gelshift assays, Western blots, DNAfootprinting, as a starting point for transcription factor puriﬁcation and more.
Mitochondrial Fractionation Kit
The Mitochondrial Fractionation Kit
isolates highly enriched mitochondrial

12 U.S.
Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
Email: sales@activemotif.com

and cytosolic fractions from mammaliancells and tissues, simplifying the study ofprotein translocation events that occur
during apoptosis and in many signaltransduction pathways. The Kit’s highquality reagents and optimized protocol
eliminate cross-contamination and produce high yields of properly segregatedmitochondrial and cytosolic fractions.

Protein Quantiﬁcation & Normalization
After you have prepared your samples,
it is also important to either quantitate
your samples or normalize them fordownstream use. Active Motif’s ProStain™
Protein Quantiﬁcation Kit is a sensitive
ﬂuorescent-based assay that offers
superior detection limits over traditional
protein assays such as Bradford, while
also offering resistance to many contaminants. As an alternative to a protein
assay, the GAPDH Whole-cell Normalization Kit can be used to normalize protein
samples, as GAPDH is constitutively expressed at high levels in almost all tissues.

Product

FormatCatalog No.

Nuclear Extract Kit

100 rxns
400 rxns

40010
40410

Mitochondrial Fractionation Kit

100 rxns

40015

GAPDH Whole-cell Normalization Kit

96 rxns

48007

ProStain™ Protein Quantiﬁcation Kit

100 rxns

15001

THE NEWSLETTER OF ACTIVE MOTIF — May 2006 • volume 7 • number 2

PRSRT STD
US POSTAGEPAID
PERMIT #236

92054