THE NEWSLETTER OF ACTIVE MOTIF — January 2007 • volume 8 • number 1

ChIP-IT™ Express

magnetic beads for a faster,
more streamlined ChIP method
2 NEW: Chromatin Assembly Kit – Test Regulation ofYour DNA in its Native Form

IN
THIS
ISSUE

3 ChIP-IT™ Express: Magnetic Beads Make Chromatin
Immunoprecipitation Easy, Saving You Hours of Time

7 Deliver Functional Proteins Directly into Living Cells
8 A Complete Solution for Transcription Factor
Analysis

4 Investigate SUMOylation with Efﬁcient SUMOlink™

9 Clone Your Target Once, Then Easily Label It with
Multiple Tags

4 MethylDetector™ – Fast, Reproducible DNA
Methylation Analysis

10 FACE™: Quantify Phospho-Proteins Without
Making Lysates

5 Multiple Methods to Study Nuclear Receptor Activity

11 Chromeo™ Dyes: Excellent Fluorescent Labels for
Bioanalysis

6 NEW: RapidReporter® – Double Destabilization andGaussia Luciferase Improve Response & Sensitivity

12 NEW: Rapid Staining of Live or Fixed Cells

Chromatin Assembly

New: Chromatin Assembly Kit – Test Regulation of Your DNA in its Native Form
The new Chromatin Assembly Kit enables you to generate chromatin in vitro
from your linear or supercoiled DNA. It yields chromatin that closely mimics
natural in vivo chromatin, so you can discover which histone modiﬁcations
and associated proteins are crucial to regulation of your target sequence.
It’s easy to generate chromatin
Now you can investigate regulation ofyour gene of interest in its native form
by assembling it into chromatin withthis easy in vitro Chromatin Assembly
Kit. This kit includes all the recombinant
proteins, core histones, buffers and ATPutilizing factors to generate chromatinfrom your DNA sequence and also toverify successful assembly. High-quality
chromatin with more than 6 regularly
spaced nucleosomes is made by adding the supplied components to 1 µg ofyour linear or supercoiled DNA, thenincubating for 4 hours. A simple partial
enzymatic digestion of the resulting
chromatin reveals the ordered spacing of
nucleosomes (Figure 1).
M

1

2

3

4

Figure 1: Enzymatic digestion of assembled chromatin.
Chromatin assembled from 1 µg samples of circular DNA(Lanes 1 & 2) and linear DNA (Lanes 3 & 4) were digested
for 2 and 4 minutes, respectively, deproteinated, phenol/
chloroform extracted and run on an agarose gel. Eachsample type resulted in regularly spaced nucleosomes.

Chromatin Assembly Kit advantages• Generate chromatin from linear orsupercoiled DNA• ATP-dependent method results
in an extended array of regularly
spaced nucleosomes• Easy protocol, simply incubate thesupplied components with your DNA• Excellent substrate for gene
regulation experiments- + p53 protein

p53 chromatin

DNA control
Figure 2: In vitro transcription of assembled chromatin.
Chromatin was incubated with p300 and acetyl-CoA in
the presence (+) or absence (-) of p53 at 30°C for
30 min. HeLa nuclear extract and NTPs (32P-labeled CTP)
were then added, along with a control DNA template,
to initiate transcription. Transcription was p53-dependent.

Why bother to make chromatin?
When DNA sequences are assembled
into chromatin by ATP-dependent
factors, the resulting structure closely
resembles the natural chromatin conﬁguration. DNA that is in a bare or unassembled state often can not reveal themechanism of transcriptional activation
or repression with the associated factors
and relevant histone modiﬁcations. However, properly assembled chromatin with

Product

FormatCatalog No.

Chromatin Assembly Kit

10 rxns

53500

2

regularly ordered nucleosomes is an excellent substrate for subsequent assays
such as in vitro transcription assays (Figure 2), chromatin immunoprecipitation(ChIP) (Figure 3), and histone acetyltransferase (HAT) assays.

Figure 3: ChIP of in vitro assembled chromatin.
Chromatin was incubated with p300 and acetyl-CoA in
the presence (+) or absence (-) of p53 at 30°C for
30 min. ChIP was then performed with p53 antibody.
Pull-down and Input DNAs were then PCR amplified
with primer pairs surrounding the p53-binding site. Pull
down of chromatin by p53 antibody was p53-dependent.

What’s in the kit?
The kit includes recombinant h-NAP-1
chaperone protein, ACF assembly complex, HeLa core histones, and the buffers
and enzymes necessary to generateassembled chromatin from your input
DNA. Control Supercoiled DNA is also
provided, and all of the kit components
have been quality control tested andproven. To verify that your chromatin
assembly reaction has been successful,reagents are provided to perform aneasy partial enzymatic digestion to visualize the regular spacing of nucleosomesby agarose gel electrophoresis (Figure 1).
Try it today!
With Active Motif’s Chromatin Assembly
Kit, you can easily assemble chromatin
on your sequence of interest to investigate gene regulation in a context thatclosely resembles in vivo chromatin. For
more information, give us a call or visit
us at www.activemotif.com.

Toll Free — 1 877 222 9543

January 2007 • volume 8 • number 1

Chromatin Immunoprecipitation

ChIP-IT™ Express: Magnetic Beads Make Chromatin
Immunoprecipitation Easy, Saving You Hours of Time
Active Motif’s ChIP-IT™ Express kits use magnetic protein G-coated beads to makechromatin immunoprecipitation (ChIP) faster and easier than traditional methods,
which use agarose beads. Magnetic beads have made it possible to streamline theprotocol and optimize buffers so time-consuming steps have been eliminated or
reduced. With ChIP-IT Express, you can get results in half the normal time withmuch less sample manipulation. And, ChIP-IT Express makes it easy to perform
ChIP on many samples at the same time (Figure 1).
ChIP-IT Express advantages• No pre-clearing, blocking or DNA
puriﬁcation• Simple wash steps improve consistency• High throughput compatible• Dramatically reduced hands-on-time
Magnetic power
ChIP-IT Express Kits include a strong bar
magnet which can be adapted to workwith any tip box or rack of your choice(Figures 1 and 2). Other commerciallyavailable racks like those from Ambion
and Promega can also be used.
ChIP-IT Express improved method
Not only does ChIP-IT Express save you
time, but the simple method generates
more consistent results than traditional
ChIP. The ChIP-IT Express magnetic beads
have much less background than agarose
beads, and this means pre-clearing andblocking steps are no longer necessary.
The magnetic pull-down occurs in justseconds, and re-formulated buffers allow steps to be combined and DNA

Figure 1: Multiple-sample ChIP using ChIP-IT Express.
Washing magnetic beads is fast and easy because thepellet forms against the side of the tube in seconds.
This makes it possible to ChIP multiple samples in
8-well PCR tubes using a multi-channel pipettor.

puriﬁcation to be eliminated. ChIP-IT
Express is available in both sonicationand enzymatic shearing formats.

Figure 2: Use of a standard tip box for magnetic ChIP.
ChIP-IT Express Kits include a strong bar magnet thatcan be used to convert tip boxes into a magnetic standfor washing the protein G-coated magnetic beads.

Ultimate controls ensure successInterpreting ChIP results can be difﬁcult,so Active Motif provides a complete
set of controls to help you understandStart your ChIP today
Active Motif’s ChIP-IT products provide
you with everything you need to makeyour ChIP experiments easier and yourresults more reproducible. No ChIP
method is faster than ChIP-IT Express,
and the validated reagents and controls
help ensure success. For more complete
information and to see what else is new,please give us a call or visit us online atwww.activemotif.com/chip.

Product

FormatCatalog No.

ChIP-IT™ Express

25 rxns

53008

ChIP-IT™ Express Enzymatic

25 rxns

53009

ChIP-IT™ Protein G Magnetic Beads

25 rxns

53014

ChIP-IT™ Control Kit - Human

5 rxns

53010

ChIP-IT™ Control Kit - Mouse

5 rxns53011

Ready-to-ChIP HeLa Chromatin

10 rxns

53015

www.activemotif.com

your results and troubleshoot yourassays. To provide you with controlsthat are appropriate for your research,
we removed the human-only controls
from ChIP-IT Express Kits and now offer
human and mouse ChIP-IT Control Kits
separately. These provide positive andnegative control antibodies and speciesspeciﬁc primer sets, PCR buffer and aconvenient 10X DNA loading dye so yourPCR reactions are gel-ready. All reagentsare quality control tested and validated
to ensure your ChIP assay is working
properly. In addition, we offer convenient Ready-to-ChIP HeLa Chromatin, so
you can be certain that the only variablein validating a new antibody for ChIP
applications is the antibody itself.

3SUMOylation & DNA Methylation

Investigate SUMOylation with Efﬁcient SUMOlink™
Active Motif’s SUMOlink™ Kits provide a simple, effective method for generating
SUMOylated proteins in vitro. These fast, efﬁcient and robust assays contain all
the necessary reagents for SUMOylation of target proteins, and include positive
and negative controls that help ensure your success.
The SUMOlink method
SUMOlink™ Kits enable you to easily
perform and detect post-translational
modiﬁcations by SUMO (small ubiquitin-like modiﬁer). With SUMOlink, you
simply add the assay components to a
microcentrifuge tube with your protein
of interest. After a 3-hour incubation,
the reaction is stopped and results canbe analyzed by Western blot (Figure 1).
With the kit’s p53 antibody and either
SUMO-1 or SUMO-2/3 antibodies, you
can easily see the extent to which yourtarget protein has been SUMOylated.

Everything you need to study SUMO
The kits contain E1 activating and E2
conjugating enzymes along with wildtype and mutant SUMO-1 (SUMO-1 Kit)
or SUMO-2 and -3 proteins (SUMO-2/3
Kit). Antibodies for SUMO-1 or SUMO2/3 modiﬁcations, as well as control p53
protein and antibody, are included. For
complete information, please give us acall or visit us at www.activemotif.com.

MW

1

2

3

1

2

3

98
64
50
36
30
16
p53 antibody

SUMO-1 antibody

Figure 1: Speciﬁc SUMO-1 labeling of p53 by SUMOlink.
Western blot analysis of in vitro SUMOylation of p53
protein by wild-type and mutated isoforms of SUMO-1:
The two Western blots were incubated with p53 antibody(1:5000 dilution) and SUMO-1 antibody (1 :4000 dilution)
Lane 1: Wild-type SUMO protein conjugation reaction.
Lane 2: Mutated SUMO protein conjugation reaction.
Lane 3: No p53 control protein used in conjugation.

Product

FormatCatalog No.

SUMOlink™ SUMO-1 Kit

20 rxns

40120

SUMOlink™ SUMO-2/3 Kit

20 rxns

40220

MethylDetector™ – Fast, Reproducible 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).

4Advantages• 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

Toll Free — 1 877 222 9543

January 2007 • volume 8 • number 1

Nuclear Receptor Assays

Multiple Methods to Study Nuclear Receptor Activity

Monitor ligand activation with NR Peptide
Studying the agonist/antagonist effects
of drug targets is important for nuclear
receptor-targeted drug discovery.
Active Motif’s NR Peptide ELISAs capture
ligand-activated nuclear receptor andcan be used with both cell extracts andproteins. Each NR Peptide ELISA Kit
provides a 96-well plate coated witha Capture Peptide that includes theconsensus-binding motif of the nuclear
receptor’s co-activator. Addition ofsample results in binding of ligand-activated nuclear receptor to the Capture
Peptide. Each well is then incubated witha primary antibody speciﬁc for the nuclear receptor of interest, followed by an
HRP-conjugated secondary antibody anddeveloping solution to provide an easily
quantiﬁed readout. This enables you
to quickly and quantitatively measure
the agonist/antagonist effects of target
compounds on the binding of ligandactivated nuclear receptors (Figure 1).

Assess DNA-binding activity of NRs
Inappropriate nuclear receptor signalingis associated with numerous diseases
including cancer, asthma and arthritis.
This makes NRs promising drug targets.
Because the end point of nuclear receptor activation is DNA binding, monitoring changes in the DNA-binding activityof a target nuclear receptor is an ideal
biomarker. Active Motif’s TransAM™ Kits
provide an innovative alternative byusing a combination of DNA binding andantibody detection to give a speciﬁc,quantitative readout of DNA-binding activity from all sample types (see page 8).
Quantify total NR with Sandwich ELISAs
In order to fully examine activation of agiven nuclear receptor, it is important tobe able to quantify its total levels withina sample. NR Sandwich ELISAs offer asimple, rapid method to quantify the
total amount of nuclear receptor proteinpresent in both cell and tissue samples.
Product

Figure 1: ERα agonism/antagonism dose-response curves.
Nuclear extracts from the breast cancer cell line MCF-7
are incubated in wells of the NR Peptide ELISA ER platein the presence of 100-fold serial dilutions (10-6 to 10-14 M)of the agonist compounds diethylstilbestrol (DES) andestradiol, and the antagonist compounds tamoxifen andclomiphene. Only ligand-activated ER can bind to the
Capture Peptide immobilized in the plate.

www.activemotif.com

NR Sandwich Kits utilize the Sandwich
ELISA-based method that improves overother methods used to study proteins,
such as Western blotting. The 96-wellformat is convenient and sensitive,
with only a minimal amount of material
required to give quantitative readout of
nuclear receptor levels (Figure 2).
AR detection (OD 450nm)

Active Motif offers a variety of nuclear receptor analysis tools that makestudying nuclear receptor proteins both faster and more accurate than using
traditional methods. Active Motif has products for monitoring DNA-binding
activity, activation state, protein level or agonist/antagonist effects.

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

LNCaP (AR+ve)
PC-3 (AR-ve)
DU 145 (AR-ve)

10

5

2.5

1.2

0.62

extract/well (µg)

Figure 2: Monitoring expression levels of AR.
Different amounts of nuclear extracts from threehuman prostate cancer cell lines, LNCaP, PC-3 and DU
145, were analyzed for levels of AR protein using the NR
Sandwich AR Kit.

A host of NR antibodies & proteins, too
In addition to its assays, Active Motif
offers over 200 highly characterized
antibodies to transcription factors andnuclear receptors, as well as recombinant nuclear receptor proteins that are
ideal for use as positive controls and inin vitro screening. Contact us today formore complete information on any of
our tools for studying nuclear receptors.
FormatCatalog No.

NR Peptide ERα

1 x 96-well plate5 x 96-well plates

49096
49596

NR Peptide ERα Chemi

1 x 96-well plate5 x 96-well plates

49097
49597

NR Sandwich AR

1 x 96-well plate5 x 96-well plates

49196
49696

NR Sandwich ERα

1 x 96-well plate5 x 96-well plates

49296
49796

NR Sandwich PR

1 x 96-well plate5 x 96-well plates

49396
49896

TransAM™ ER

1 x 96-well plate5 x 96-well plates

41396
41996

TransAM™ GR

1 x 96-well plate5 x 96-well plates

45496
45996

TransAM™ PPARγ

1 x 96-well plate5 x 96-well plates

40196
40696

5

Luciferase Assay

New: RapidReporter® – Double Destabilization
and Gaussia Luciferase Improve Response & Sensitivity
Active Motif’s new RapidReporter® is a patented* method that provides a fasterand more pronounced response to stimulation than other reporter gene assays.
Because RapidReporter vectors include destabilization elements for both theluciferase protein and its mRNA, the assay yields more accurate kinetic and drugconcentration-dependent responses. This vastly improves the monitoring oftranscription factor activation and the detection of active compounds duringhigh-throughput screening. In addition, RapidReporter vectors feature Gaussia
luciferase, which is brighter and much smaller than ﬁreﬂy and Renilla luciferases.
Because of their simplicity and versatility,
reporter gene assays are an important
tool for studying signal transduction
pathways and gene expression. In suchassays, a promoter or enhancer element
is cloned into a vector upstream of a reporter gene, commonly luciferase. Aftertransfection, the cells are stimulated toinduce transcription from the cloned
promoter element. The assay uses theincrease in luciferase protein levels as ameasure of transcriptional activity.
Standard luciferase assays, however, arelimited by the fact that basal activity of
the cloned promoter results in accumulations of luciferase mRNA and protein. The
slow clearance rate of these pre-existing
reporter molecules substantially delays
and dilutes the measurable response tostimulation. This is because the short,
rapid increase in luciferase that occurs in
response to stimulation has proportionally little impact on the high steady-statelevels already present. As a result, withstandard reporter gene assays, transientor relatively minor effects are hidden andkinetic assays are inaccurate.
Double destabilization is the key
To solve the problem of long half-life
proteins, some assays incorporate a
protein-destabilizing element in the

6vector, causing the luciferase to degrade
more rapidly. Destabilizing the reporter
protein, however, only partly addresses
the problem as reporter clearance rates
are also dependent on the half-lifeof the reporter mRNA. As long as thepre-existing reporter mRNA remains
intact, it continues to produce newreporter protein. To address this issue,
the patented RapidReporter® method
utilizes vectors that include both protein
AND mRNA destabilizing elements. This
makes the assay much more responsive
to both increases and decreases in transcriptional activity. Thus, RapidReporter
provides a more accurate measurementof the actual transcriptional activity of
the cloned promoter.

RapidReporter advantages• Increased magnitude of response
provides better separation of active
vs. inactive compounds, which will
reduce false positives• Effect of weak to moderate stimulations, which are not found by otherassays, are easily detected• Faster response time enables thedetection of transient effects andof drugs that decompose rapidlyYour choice of stringency
RapidReporter vectors are offered in
two different stringencies. pRR-High ishighly destabilized, while pRR-Low has
fewer elements, which is appropriate
when studying weaker stimulation.
Pre-made vectors & complete kits
In addition to empty RapidReporter
vectors, Active Motif offers vectorsthat contain widely studied promoters.
All vectors are available separately or incomplete assay kits, which also include
a positive control vector, Lysis and Assaybuffers, and a substrate optimized for
Gaussia luciferase. For more complete
information on this exciting new technology, please give us a call or visit our
website at www.activemotif.com.

Product

FormatCatalog No.

RapidReporter® Gaussia Luciferase Assay

100 rxns
1000 rxns

33001
33002

RapidReporter® pRR-High vector
RapidReporter® pRR-High Assay

10 µg
100 rxns

33003
33004

RapidReporter® pRR-Low vector
RapidReporter® pRR-Low Assay

10 µg
100 rxns

33005
33006

RapidReporter® pRR-High-CRE vector
RapidReporter® pRR-High-CRE Assay

10 µg
100 rxns

33007
33008

RapidReporter® pRR-High-NFκB vector
RapidReporter® pRR-High-NFκB Assay

10 µg
100 rxns

33009
33010

* RapidReporter is covered under U.S. Patent No. 7,157,272 and various other patents worldwide, and issold under license granted by GeneStream Pty Ltd. Purchasers are subject to a Limited-use License;
please contact Active Motif’s Technical Services or download a product manual from our web site
for details. RapidReporter is a registered trademark of GeneStream Pty Ltd.

Toll Free — 1 877 222 9543

January 2007 • volume 8 • number 1

Protein Delivery

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.

7

Transcription Factor ELISAs

A Complete Solution for Transcription Factor Analysis
TransAM™ Kits make it straightforward to measure the activity of entire families oftranscription factors in one simple experiment that takes less than 5 hours. Plus,
TransAM Kits can be used on all sample types, including cell lines, primary isolates
and tissues, giving you unsurpassed ﬂexibility.
Save time and money
Transcription factor activity is often
studied using Electrophoretic Mobility
Shift Assays (EMSA), immunoblotting andreporter gene assays. But, these methods are time consuming and, at best,
provide only semi-quantitative results.
Moreover, determining the activity oftranscription factor subunits by thesemethods is costly in terms of time andmoney, and may not even be possible.
TransAM™ Kits, however, use a uniqueplate-based format to capture activatedtranscription factors, which are analyzed
using antibody speciﬁc to your isoform
of interest (Figure 1). As a result, levels of
entire transcription factor families canbe measured in a matter of hours, saving
you time and money.
The TransAM advantage• Up to 100-fold more sensitive thangelshift assays• Eliminates the use of radioactivity
and the need to run gels
• Results in less than ﬁve hours• Colorimetric readout enables easy,
quantitative analysis• 96-stripwell format enables both
high and low throughput

Functional assay for all sample types
Many commonly used transcription
factor analysis tools, such as gelshift andreporter assays, either lack sensitivity,
or cannot be used with primary material, such as tissues or peripheral bloodmononuclear cells (PBMCs). This canmake comparing primary and cell model
data difﬁcult and lead to inaccuracies.
TransAM, however, is up to 100-foldmore sensitive than gelshift assays andcan be used with cell or nuclear extract
to functionally monitor transcription
factor activation in any sample type.
Consequently, TransAM is the mostwidely cited DNA-binding ELISA available
today. So, why not give us a call or visit
www.activemotif.com/transam to ﬁnd
out more, including getting the list of
the 100’s of papers that cite TransAM.

Widest selection of targets and formats
Active Motif is proud to offer the broadest selection of transcription factorbinding ELISAs, including kits for NFκB
(p65, p50, p52, c-Rel and RelB), AP-1
(c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 andFra-2), STAT (1α, 3, 5A and 5B), PPARγ,
HIF-1, NFATc1, C/EBP and many others(Table 1). In addition, TransAM Kits areavailable in either colorimetric or ultrasensitive chemiluminescent* readout.
And for the ultimate ﬂexibility, TransAM
Flexi NFκB Kits let you use any capture
oligo you choose.
Add nuclear
extract

Add primary
antibody

Add anti-IgG
HRP conjugate

1 hr.

1 hr.

Cell extractcontaining activatedtranscription factor

Add developing
and stop solution

1 hr.

Oligonucleotide
coated plate(Flexi Kits enable any
oligo to be coated)

Figure 1: Flow chart of the TransAM process.

Table 1: TransAM™ Product Line
TransAM™ AP-1 Family

TransAM™ AML-1/Runx1

TransAM™ C/EBP α/βTransAM™ HNF-1

TransAM™ Flexi NFκB p65

TransAM™ GATA Family

TransAM™ AML-3/Runx2

TransAM™ CREB & pCREB

TransAM™ IRF-3

TransAM™ NFκB p65*

TransAM™ HNF Family

TransAM™ AP-1 c-Fos

TransAM™ Elk-1

TransAM™ MEF2

TransAM™ Oct-4

TransAM™ MAPK Family

TransAM™ AP-1 c-Jun

TransAM™ ER

TransAM™ MyoD

TransAM™ p53

TransAM™ Flexi NFκB Family

TransAM™ AP-1 FosB

TransAM™ FKHR (FOXO1/4)

TransAM™ NF-YA

TransAM™ PPARγTransAM™ NFκB Family

TransAM™ AP-1 JunD

TransAM™ GATA-4

TransAM™ NFATc1

TransAM™ Sp1 & Sp1/Sp3

TransAM™ STAT Family

TransAM™ ATF-2

TransAM™ GR

TransAM™ Flexi NFκB p50

TransAM™ STAT3

TransAM™ c-Myc

TransAM™ HIF-1

TransAM™ NFκB p50*

* The Original TransAM NFκB p50 & p65 Kits are offered in both colorimetric and chemiluminescent formats. TransAM Chemi Kits require the use of a luminometer.

8

Toll Free — 1 877 222 9543

January 2007 • volume 8 • number 1

Protein Labeling & Translocation

Clone Your Target Once, Then Easily Label It with Multiple Tags
Active Motif’s LigandLink™* Universal Labeling technology enables you to label
your target gene in living cells with multiple ﬂuorescent tags and just clone yoursequence into one vector. The easily changed tags make LigandLink fusion proteins
ideal for co-localization studies.
Superior and ﬂexible dye options
With LigandLink™, the gene of interestis cloned into the vector once, and thissingle clone can be labeled with different LigandLink ﬂuorescent tags. The
ﬂuorescent tags are cell permeable andhighly speciﬁc for the LigandLink fusion
protein, so there is very low background(Figure 1). And, the fusion protein is small(~18 kDa), so protein activity is less likely
to be affected than with GFP (27 kDa).
LigandLink Labels are available with both
red and green ﬂuorescent dyes (Figures 1
and 2). These labels are stable and ideal
for co-localization research.

reductase (eDHFR) gene in the LigandLink pLL-1 vector. Then, transfectthe vector into mammalian cells to
express the fusion protein. Twenty-fourhours later, label the protein of interestby adding a LigandLink label to the cell
culture medium. Depending on the cell
type, the labeled fusion protein can bevisualized in as little as 10 minutes. The
LigandLink ﬂuorescent label is a TMP(trimethoprim) ligand that speciﬁcallyand rapidly binds to eDHFR and does notinteract with mammalian proteins. This isbecause TMP is an antibiotic designed toinhibit the bacterial eDHFR gene with ahigh binding speciﬁcity (Kl ≈ 1 nm).

Figured 2: Labeling of nuclear-localized eDHFR by
LigandLink Fluorescein.
A nuclear localization sequence was cloned into pLL-1
and transfected into CHO cells. Twenty-four hours
post-transfection, 5 µM LigandLink Fluorescein wasadded to the cells for 10 minutes.

Try LigandLink today
LigandLink is an improved way to label
proteins in living cells. It is easy tochange labels by adding a different
LigandLink tag. Find out more by visitingwww.activemotif.com or calling us today.

The LigandLink method
Simply clone your gene of interest inframe with the E. coli dihydrofolate

���
����

�
���
Figure 1: 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.

LigandLink advantages• Clone once, but obtain different
functionalities by using different
LigandLink labels• Small label is unlikely to interferewith protein function• LigandLink labels are cell permeable• LigandLink does not interact withother mammalian proteins�������������������

�����

�

�
����

���

���

Figure 3: 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 one of the cell-permeable
LigandLink Labels to the medium results in rapid, specific binding of the label by the fusion protein.

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.

www.activemotif.com

9Phospho-Protein Detection

FACE™: Quantify Phospho-Proteins Without Making Lysates
Active Motif’s Fast Activated Cell-based ELISA (FACE™) Kits provide you witha simple, sensitive, cell-based method for detecting protein phosphorylation
directly in the cell, without making cell extracts or the trouble of running gels.
“freezes” the cellular state and prevents
further protein modiﬁcations. This
enables detection of the exact proteinstate in the cells at your chosen time
points, providing more accurate results.

The “in-cell” Western method
In FACE™, cells are grown in a 96-wellplate and stimulated to induce the pathway of interest. After stimulation, thecells are ﬁxed to preserve protein activation and modiﬁcation states. Each well isthen incubated with a primary antibody
speciﬁc to the protein of interest. An
antibody that recognizes all forms of theprotein is included as well as an antibody
that recognizes the phosphorylatedsite of the target protein so you cancompare the stimulation effect in yourcells. After the primary antibody incubation, an HRP-conjugated secondary isadded along with either colorimetric or
chemiluminescent developing solutions(Figure 1). The results provided are bothquantitative and reproducible (Figure 2).

“FACE Kits enable detection ofphosphorylated proteins within thecell, eliminating the need for cell
extractions, gels & Western blotting.”

Prevent unwanted sample modiﬁcations
The FACE method eliminates the needto prepare cell lysates, which greatly
reduces the possibility of introducing
undesired alterations of the protein ofinterest. FACE uses a ﬁxation step that

Phospho JNK
Total JNK

0.5
0.4

OD450 nmFACE advantages• No cell extracts or immunoblotting• Less than 2 of hours hands on time
• Total and phospho-speciﬁc
antibodies included

0.6

0.3
0.2
0.1

0

0

0.1
10
1
Anisomycin induction (µg/ml)

Figure 2: 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.

formats for over 20 different kinase andreceptor targets (Table 1). And, with the
FACE Maker Kits, you can use your own
primary and secondary antibodies todetect any target or modiﬁcation stateof interest. For complete information,
visit www.activemotif.com/face.

A variety of kits to choose from
FACE kits are available in bothchemiluminescent and colorimetric

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 1: 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.

Table 1: The 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 (Y845)

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™ HSP27 (S82)

FACE™ JAK1 (Y1022/Y1023)

FACE™ JNK (T183/Y185)

FACE™ Maker

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)

10

Toll Free — 1 877 222 9543

January 2007 • volume 8 • number 1

Fluorescent Detection

Chromeo™ Dyes: Excellent Fluorescent Labels for Bioanalysis
Active Motif’s Chromeo™ Dyes are superb labels for cell culture with theirrobust photostability, low cell toxicity and broad range of pH stability. And,
they can be used with many common excitation sources.

�������������������������

Superior ﬂuorescent properties
The Chromeo™ Dyes, Chromeo 494,
Chromeo 546 and Chromeo 642, are ideal
for any ﬂuorescent application becauseof their superior luminescent properties and broad Stokes shifts (Table 1).
Chromeo Dyes are suitable for use withmany common excitation sources such as
diode lasers, LEDs, tungsten and Xenon
arc lamps.

���������
���������

cent 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.

����������
����������

�����������������
�������������

�
���
���
���
���
�

�

��

��

��

��

�����������

��

��

��

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

55,00025

124 nmChromeo™ 546

545

561

98,8001016 nm

Chromeo™ 642

642

660

180,00015

18 nm

Product

FormatCatalog No.

Chromeo™ 494 Carboxylic Acid

1 mg
5 mg

15110
16110

Chromeo™ 494 NHS-Ester

1 mg
5 mg

15111
16111

Chromeo™ 494 Biotin

1 mg
5 mg

15112
16112

Chromeo™ 494 Streptavidin

1 mg
5 mg

15113
16113

Chromeo™ 546 Carboxylic Acid

1 mg
5 mg

15210
16210

Chromeo™ 546 NHS-Ester

1 mg
5 mg

15211
16211

Chromeo™ 546 Biotin

1 mg
5 mg

15212
16212

Chromeo™ 546 Streptavidin

1 mg
5 mg

15213
16213

Chromeo™ 642 Carboxylic Acid

1 mg
5 mg

15310
16310

Chromeo™ 642 NHS-Ester

1 mg
5 mg

15311
16311

Chromeo™ 642 Biotin

1 mg
5 mg

15312
16312

Chromeo™ 642 Streptavidin

1 mg
5 mg

15313
16313

Dye

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

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

Better for biology
Chromeo Dyes exhibit key features thatmake them better than other labels
for ﬂuorescent microscopy. Chromeo
conjugates are bright and readily soluble(Figure 1). Their brightness intensity,
limited photobleaching (Figure 2), andbroad Stokes shifts make them easy topair with other ﬂuorescent probes. In
particular, Chromeo 494 has a Stokes
shift of 124 nm that makes it an ideal
partner for multiplexing.
Chromeo capabilities
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 phosphores-

www.activemotif.com

11

Live Cell Staining

New: Rapid Staining of Live or Fixed Cells
Active Motif’s new LavaCell™ stain is a unique, non-toxic, cell permeable
dye, ideal for use in the study of cell proliferation, cell counting, chemotaxis,
cell morphology or any application that requires cellular imaging.
LavaCell™ is a small, naturally ﬂuorescentcompound with inherent properties thatmake it ideal for cell imaging:• Non-cytotoxic• Does not alter cell growth• Cell permeable• 405, 488 and 532 nm excitable• Brightly ﬂuorescent at biological pH(Figure 1)• Non-ﬂuorescent prior to cellularinternalization
Ideal for multiplexing
LavaCell has a signiﬁcant Stokes shift(488 nm ex:610 nm em), which meansthat it is ideally suited for use with othergreen-emitting ﬂuorophores that are

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

commonly used in cell biology (Figure 2).
It is also excited by a variety of common
laser sources (405, 488 and 532 nm).

Figure 1: Staining of live CHO cells with LavaCell.

Just add and image
Using LavaCell is simple; just add it toyour cell culture, wait 30 minutes andanalyze. Nothing could be easier! The
dye readily diffuses into live or ﬁxed
cells to produce bright orange ﬂuorescence on the plasma and internal membranes. And, there is no need to wash
away unbound dye. Please call or visit us
online for more complete information.

Figure 2: LavaCell and Calcein AM in live CHO cells.

Product

FormatCatalog No.

LavaCell™

200 µg15004

THE NEWSLETTER OF ACTIVE MOTIF — January 2007 • volume 8 • number 1

PRSRT STD
US POSTAGEPAID
PERMIT #236

92054