THE NEWSLETTER OF ACTIVE MOTIF — APRIL 2005 • VOLUME 6 • NUMBER 1

800000IN THIS ISSUE

Unstimulated
EGF stimulated

600000

New version lets you choose your site
The original TransAM Kits are DNA-binding
ELISAs that provide a fast, non-radioactive alternative to classical methods suchas Electrophoretic Mobility Shift Assays(EMSAs). Each kit includes a 96-well platethat is coated with a double-stranded
oligonucleotide, which contains a specific consensus-binding site. While this

New: ELISA-based Method for
Fast, Sensitive Quantification of
Activated Ras GTPase

1

New: Measure Transcription
Factor Binding at Any
DNA Sequence You Choose2

New: Simplify Chromatin
Shearing Using the Enzymatic
Shearing Kit for ChIP

3

Chariot™ Delivers Functionally
Active Proteins, Peptides andAntibodies into Live Cells

5

New: A Novel, Cell-based
Method for Monitoring
STAT Phosphorylation

7

A Complete Solution forStudying Nuclear Receptor
Protein Activity

8

New Antibodies for Chromatin
and DNA Methylation

400000200000
0

6.25

12.5
25
Cell extract (µg/well)50

Figure 1: Quantification of activated Ras in stimulated HeLa cells.
Increasing amounts of whole-cell extract from HeLa cells that had
been stimulated with 50 ng/ml of EGF for 2 minutes were assayedusing the Ras GTPase Chemi ELISA Kit.

continued on page 6

New: Measure Transcription Factor Bindingat Any DNA Sequence You Choose
Active Motif’s new TransAM™ Flexi Kits
make it possible to efficiently study thebinding of transcription factors at any
DNA-binding site you choose. These kits
are the latest version of Active Motif’s
original TransAM Kits, which have rapidly
become the method of choice for studying
transcription factor binding activity.

1

format makes it simple and convenient
to measure transcription factor bindingat this consensus site, it does not allow
you to study binding of the transcription factor at alternative sites, such as
those which contain mutations or that are
putative, unproven binding sites. The newTransAM Flexi Kits give you the flexibility
to immobilize any oligo or PCR productin the 96-well plate, while providing you
with proven antibodies and all of the othercomponents you’ll need to quantitatively
measure transcription factor binding. With
TransAM Flexi Kits, you can now study
variant transcription factor-binding sites,
analyze native promoters, confirm chromatin immunoprecipitation (ChIP) results anddetermine isoform-binding affinity.

NFκB Activation (OD450 nm)

Active Motif is pleased to introduce the
new Ras GTPase Chemi ELISA Kit. It is thefirst ELISA-based kit designed to detect andquantify activated Ras GTPase. As an ELISA,
it offers a number of advantages over existing methods. The Ras GTPase Chemi ELISA
Kit is much more sensitive than pull-down/Western methods, so you can detect even
low levels of protein, and use less sample.
In addition, ELISAs are more quantitative
than Westerns, and they eliminate the needto run and develop gels. This means you’ll
get better results faster, and with less effort. And, as the assay uses a 96-well plate
made up of 12-well strips, it is convenientand economical to run anywhere from
1 to 96 samples in a single experiment.

Activated Ras Signal (RLU)

New: ELISA-based Method for Fast, Sensitive
Quantification of Activated Ras GTPase

1

Unstimulated
TPA + CI withwild-type oligos

TPA + CI stimulated
TPA + CI withmutant oligos

0.8
0.6
0.4
0.2
0

Figure 2: Specificity of TransAM Flexi assays.
TransAM Flexi NFκB p65 assays are performed in the presence of
wild-type and mutant competitor oligonucleotides using 5 µg/well of
nuclear extract from Jurkat cells that were stimulated with TPA andcalcium ionophore (CI).

continued on page 4

www.activemotif.com

Chromatin Immunoprecipitation

New: Simplify Chromatin Shearing Using the Enzymatic Shearing Kit for ChIP
Active Motif is pleased to announce
the release of another innovative product that simplifies your chromatin
immunoprecipitation (ChIP) experiments.
The new Enzymatic Shearing Kit is your best
bet for generating chromatin suitable for
ChIP as well as other applications that study
chromatin structure with little to no optimization required – nothing could be simpler.
Successful shearing every time
In order to perform a successful ChIP
experiment, chromatin must first be sheared
to 200-1000 bp fragments. Traditionally,shearing has been performed by subjecting
the isolated chromatin to different pulses ofsonication. Although sonication can be���� ����� ����� ��
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Figure 1: Analysis of DNA sheared using the Enzymatic Shearing Kit.
HeLa cells were fixed for 10 minutes with 1% formaldehyde and thenchromatin was prepared using the Enzymatic Shearing Kit protocol.
Chromatin was sheared with the Enzymatic Shearing cocktail for
5, 10 & 15 minutes and the reaction was stopped. The sheared andunsheared chromatin samples were subjected to cross-link reversal,
treated with Proteinase K, phenol/chloroform extracted andprecipitated as described in the protocol. Samples were separated byelectrophoresis through a 1% agarose gel to assess shearing results:
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.
Lane 5: HeLa DNA treated for 15 minutes.

an effective method for shearing DNA, it
can also be time consuming and difficult to
optimize due to complications arising fromemulsification and overheating during thesonication process. Also, because the qualityof your sheared sample depends greatly
upon the quality of your sonicator, it may benecessary to purchase an expensive, “highend” sonicator to get reproducible shearing.
Because of this, Active Motif has developed
the Enzymatic Shearing Kit, which is a morerobust and user-friendly method to shear
chromatin for ChIP. The Enzymatic Shearing
Kit uses a proprietary enzymatic shearing
cocktail that quickly and easily shears DNA
into 200-1000 bp fragments (Figure 1). And,because enzymatic shearing is dependent
only on time and temperature, all of the
problems associated with sonication are
eliminated and ChIP results are improved.
How it works
Shearing chromatin with the Enzymatic
Shearing Kit is simple to perform. First, cells
are grown to confluency and then fixedusing formaldehyde, which cross-links andtherefore preserves protein/DNA interactions. A Glycine “Stop-Fix” buffer is thenadded to stop the fixation procedure andprevent over-crosslinking of the sample.
Next, the cells are lysed and the nuclei
are collected. A simple, 10-minute incubation with the enzymatic cocktail is thenperformed, the reaction is stopped and thechromatin is ready to use.
How can I get one
The Enzymatic Shearing Kit is sold separately and can be used in conjunction with
Active Motif’s ChIP-IT™ Kit buffers andcontrols. Order an Enzymatic Shearing Kit
today and simplify your shearing.

ChIP Product Line
Complete ChIP-IT Kits
Active Motif’s ChIP-IT Kit is the mosteffective product available for performing ChIP. The kit contains nearly
everything you need to perform ChIP
including a comprehensive protocol
and optimized buffers, inhibitor
cocktails, DNA purification columns
as well as positive control primers andantibodies, all of which have beenvalidated in actual ChIP experiments.
Pre-validated ChIP antibodies
Finding an antibody that has beenvalidated in ChIP experiments can bedifficult. As specialists in transcription
factor research, Active Motif offers alarge number of antibodies, which areactively being validated for use in ChIP.
Below is a list of antibodies that havebeen positively tested in ChIP, which
are ideal when used with our ChIP-IT
w/o controls kit. Be sure to keep up todate on new ChIP-validated antibodies
by visiting our website.
Antibody

Cat. No.

AML-1/Runx1 pAb

39000

AP-2 pAb

39304

c-Jun pAb

39309

C/EBPα pAb

39306

DNMT1 mAb

39204

DNMT3A mAb

39206

DNMT3B mAb

39207

E2F-1 pAb

39313

E2F-6 mAb

39509

GATA-1 pAb

39025

HBP-1 mAb

39511

HDAC3 pAb

40968

HDAC4 pAb

40969

HDAC5 pAb

40970

HDAC6 pAb

40971

IRF-3 pAb

39033

JunB pAb

39326

JunD pAb

39328

p53 pAb39334

Pax-5 pAb

39336

Product

FormatCatalog No.

PPARγ pAb

39338

Enzymatic Shearing Kit

10 rxns

53005

RNA pol II mAb

39097

ChIP-IT™ Kit

25 rxns53001

Sp1 pAb

39058

53004

TRF2 pAb

39223

ChIP-IT™ w/o controls

2

25 rxns

Toll Free — 1 877 222 9543

APRIL 2005 • VOLUME 6 • NUMBER 1

Protein Transfection

Chariot™ Delivers Functionally Active Proteins, Peptides and Antibodies into Live Cells
Chariot™ is Active Motif’s patented* protein
delivery reagent that efficiently transports
biologically active proteins, peptides andantibodies directly into cultured mammalian cells in less than two hours. The cells
can be assayed immediately after delivery
to determine the effects of the introduced
material, without the need for fixing. This
makes Chariot ideal for a variety of functional studies in living cells.

? How does Chariot work?
Chariot is a peptide that forms a noncovalent complex when combined witha purified protein, peptide or antibody.
When added to cells, the complex is rapidlyinternalized. After delivery, the complex
dissociates in a process called “decaging”,
leaving the macromolecule biologically active and free to proceed to its cellular target. One advantage of the complex is thatit stabilizes the macromolecule, protecting
it from degradation during internalization.
It also eliminates the need to make fusion
proteins or perform chemical coupling.
Chariot delivery is also serum independent,
which gives you the flexibility to culture yourcells in whichever type of media you prefer.
Chariot work with primary? Doescells or even in vivo?
Chariot effectively delivers into primarycells, something that most other delivery
systems cannot do. Most notable is the useof Chariot on hard-to-transfect primaryneurons (Jurney, W. et al. (2002) Journal of
Neuroscience 22: 6019-602) without any
signs of cytotoxicity or rejection. Chariot
has also been shown to deliver proteins invivo (Aoshiba, K et al. (2003) Am J Respiratory Cell & Molecular Biology. 28: 555-562).

?

What about protein functionality?

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 the Chariot-galactosidase complex, demonstrating successful delivery offunctional β-galactosidase (Figure 1).

Log on to www.activemotif.com/chariot todownload the protocol as well as a current
list of publications that cite Chariot.
there any size limitation on the? Isproteinsthat Chariot can deliver?
There is one limitation; peptides below 12
amino acids are too small for Chariot to efficiently deliver. However, we have not founda maximum size limitation. The key to remember is that the larger the protein or antibody,the longer it takes to get in; but it will get in.

Figure 1: Chariot delivery of β-galactosidase.
One µg of a 119 kDa subunit of β-galactosidase was complexed with
Chariot for 30 minutes and delivered into HeLa cells. Cells were fixedand stained with X-gal 2 hours post-delivery.

are researchers using? WhatChariot for?
We have an ever growing list of citations
from researchers who have used Chariot intheir published research. Chariot is becoming the number one choice for protein,
peptide and antibody delivery into bothprimary and transformed cell lines. Peluso
et al transfected a peptide and an antibody
into granulosa cells to study the PKCdDependent Pathway (Endocrinology. (2001)
Vol. 142(10): 4203-4211).
Another recent publication shows theusage of Chariot in primary T cells to study
the effects of a novel protein on T cell
activation (Gorska, M. et al. (2004) J Exp.

Product

FormatCatalog No.

Chariot™

25 rxns
100 rxns

30025
30100

β-Galactosidase Staining Kit

75 rxns35001

www.activemotif.com

Med. 199(3): 369-379). And Ikari et al.
used Chariot to study up-regulation of
sodium-dependent glucose transporter byinteraction with Heat Shock protein 70 inepithelial cells (JBC. (2002) Vol. 277, No. 5.
33338-33343).

Chariot delivery use the? Doesendosomal pathway?
No, Chariot delivery is independent of theendosomal pathway. Temperature studies
performed at 37°C and at 4°C show nearly
identical results. This demonstrates that
Chariot is independent of the endosomal
pathway, as it is shut down at 4°C. Because
endosomal delivery can modify proteins, thisis a further advantage of Chariot delivery.
guarantee that my protein? Dowillyoube deliveredby Chariot?
Complex formation depends on the sizeand charge of the protein, and delivery of
the complex is influenced by your specificcell line’s membrane. Because of thesevariabilities, there are some proteins and/or
cell lines that are not transfected efficiently
by Chariot. However, the β-galactosidase
protein is provided as a positive control
with Chariot to make it easy for you to
optimize delivery conditions for your cellline using our own gold standard.
* Technology covered under U.S. Patent No. 6,841,535, which is licensed
to Active Motif. Purchase includes rights for research use. Other-use
licenses available; contact technical services for details.

3DNA-binding ELISAs

continued from page 1 — New: Measure Transcription Factor Binding at Any DNA Sequence You Choose
The TransAM Flexi method
In the TransAM Flexi method, a biotinylated
oligo or PCR product, which contains thetranscription factor-binding site of choice,is incubated with nuclear extracts that havebeen treated to activate the transcription
factor. The samples are then transferred
to a 96-well, streptavidin-coated plate,where the biotinylated oligonucleotide iscaptured. A primary antibody specific forthe activated transcription factor is thenadded to the individual wells, followed by
an HRP-conjugated antibody and developing reagent. The plate is then read ona spectrophotometer, which provides aquantitative, colorimetric readout oftranscription factor activation (Figure 3).
Assay a variety of sequences for moreaccurate disease research
Historically, it has only been possible tomeasure the binding activity and affinityof a transcription factor to non-consensus
sequences, such as putative or polymorphic
sites, using either reporter gene constructs
or radioactive electrophoretic mobility
shift assays (EMSAs). Unfortunately, theseapproaches are time consuming, lacksensitivity and require significant validation
before they can be used. The new TransAM
Flexi Kits, however, make sequence-specifictranscription factor studies more straightforward than ever before. This is because
every TransAM Flexi Kit is supplied as acomplete solution of validated antibodies,
controls and preparation & reaction buffers,
so all you need to do is decide how manydifferent sequences you want to test.

Add primary
antibody

Add the extract-DNA
cocktail to plate

Add anti-IgG
HRP conjugate

1 hr.

1 hr.

Add developing
and stop solution

1 hr.

Streptavidin
coated plate

Incubate biotinylated DNA with cell extractcontaining activated transcription factor
Figure 3: Flowchart of the TransAM Flexi procedure.

Proven antibodies to ensure your success
As in chromatin immunoprecipitation,transcription factor-binding assays require
the use of highly specialized antibodies.
Developing antibodies with the properties needed for use in TransAM is no easytask. They must be able to recognize thetarget protein when it is bound to DNA,and they must bind both specifically andwith high affinity. To ensure that you get
the highest quality results, every TransAM
antibody has been extensively tested forsensitivity and specificity (Figure 2, page 1).

to analyze the ability of a transcription
factor to bind to any sequence, they offer
a fast and convenient tool to confirm thatyour ChIP results are valid.
TransAM Flexi advantages• Flexible assay – enables analysis ofbinding at any DNA sequence• Reproducible – proven antibodies
and reagents ensure your results• Sensitive & quantitative – ELISAs
provide better results than Westerns• Fast – have results in less than 5 hours• Versatile – assay cell or tissue samples

Confirm your ChIP results
ChIP is fast becoming an invaluable tool forthe analysis of gene promoter regulation.
However, there are currently no convenient
tools that can be used to confirm if thetranscription factor of interest is capableof binding to the promoter being studied.
Because TransAM Flexi Kits make it possible

Study your chosen sequences today
TransAM Flexi Kits make it fast and easy for
you to study transcription factor bindingat any DNA sequence you choose. Kits for
NFκB p50 and p65 are now available, withmore on the way. Try one today!

TransAM™ Product Line
TransAM™ AP-1 Family

New: TransAM™ Flexi NFκB p50

TransAM™ ATF-2

TransAM™ GR

TransAM™ NFκB p50*

TransAM™ GATA Family

New: TransAM™ Flexi NFκB p65

TransAM™ c-Myc

TransAM™ HIF-1

TransAM™ NFκB p65*

TransAM™ HNF Family

New: TransAM™ AML-1/Runx1

TransAM™ C/EBP α/βTransAM™ HNF-1

TransAM™ Oct-4

TransAM™ IRF Family

TransAM™ AML-3/Runx2

TransAM™ CREB & pCREB

TransAM™ IRF-3

TransAM™ p53

TransAM™ MAPK Family

TransAM™ AP-1 c-Fos

TransAM™ Elk-1

TransAM™ MEF2

TransAM™ PPARγTransAM™ NFκB Family

TransAM™ AP-1 c-Jun

TransAM™ ER

TransAM™ MyoD

TransAM™ Sp1 & Sp1/Sp3

TransAM™ STAT Family

TransAM™ AP-1 FosB

TransAM™ FKHR (FOXO1)TransAM™ NF-YA

TransAM™ STAT3

TransAM™ AP-1 JunD

TransAM™ GATA-4

TransAM™ NFATc1* The Original TransAM NFκB p50 & p65 Kits are offered in both colorimetric and chemiluminescent formats. TransAM Chemi Kits require the use of a luminometer.

4

Toll Free — 1 877 222 9543

APRIL 2005 • VOLUME 6 • NUMBER 1

Cell-based ELISAs

New: A Novel, Cell-based Method for Monitoring STAT Phosphorylation

The importance of STATs
The new FACE STAT Kits make it easy to
study phosphorylation of STAT proteins.
STATs (signal transducers and activators oftranscription) are latent transcription factors that are activated by phosphorylation
via tyrosine kinases. Because the disruptionof STAT signaling blocks neoplastic transformation, inhibitors of STAT proteins havebecome important drug candidates for thetreatment of cancer.
The FACE method
In the FACE method, cells are cultured in
96-well 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).
FACE Kits also contain a primary antibody
for the native non-modified protein, so
you can monitor both native and activatedprotein levels in the same experiment.
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(Figure 2)

www.activemotif.com

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.

Reproducible, scaleable assay
Typically, phospho-specific analyses are
performed on small sample numbers
because the classical methods of study,
such as Western blot and in-gel kinase
assays, are both labor intensive and timeconsuming. This forces you to run multiple
experiments on different days and then tocompare the results. However, because thevariability of these methods is high andinduction of phosphorylation is typicallylow, this can lead to statistically poor data.

1200000
STAT Chemi signal (RLU)

Fast Activated Cell-based ELISA (FACE™) Kits
provide a simple, innovative alternative toclassical methods for monitoring protein
phosphorylation. With FACE, modificationspecific analysis can be performed without
time-consuming cell extractions, gel electrophoresis or membrane blotting.

Fortunately, FACE Kits make running more
samples as simple as adding an extra tip toyour multi-channel pipettor. And, because
FACE Kits are highly reproducible, yourresults will be more statistically relevant.
Try FACE Kits today
Current FACE Kits available are listed below.
For complete information, please give usa call, return the enclosed reply card or
download the product manual and productcitation list at www.activemotif.com/face.

Uninduced
Induced1000000

Figure 2: Measuring phosphorylated and total STAT.
NIH/3T3 cells were cultured in 96-well plates and serumstarved for 16 hours. Cells were then treated with 50 ng/ml
PDGF for 5 minutes and fixed. Total and phospho STAT2,
STAT4 and STAT6 were each assayed in triplicate using thephospho and total STAT antibodies included in the FACE
STAT Kits. Data was plotted after correction for cell number(performed through use of Crystal Violet).

8000006000004000002000000Total- Phospho- Total- Phospho- Total- PhosphoSTAT2 STAT2 STAT4 STAT4 STAT6 STAT6FACE™ Product Line
FACE™ AKTFACE™ ATF-2

FACE™ Bad

FACE™ c-Jun (S63)

FACE™ c-Jun (S73)

FACE™ c-Src

FACE™ EGFR (Y992)

FACE™ EGFR (Y1173)

FACE™ ErbB-2 (Y877)

FACE™ ErbB-2 (Y1248)

FACE™ ERK1/2

FACE™ FAKFACE™ FKHR (FOXO1)FACE™ GSK3β

FACE™ JAK1FACE™ JNKFACE™ MEK1/2

FACE™ NFκB Profiler

FACE™ p38

FACE™ PI3 Kinase p85

FACE™ STAT2

FACE™ STAT4

FACE™ STAT6

5

Ras Activation Assaycontinued from page 1 — New: ELISA-based Method for Fast, Sensitive Quantification of Activated Ras GTPase
GTPases are important regulators
Small GTPases, also known as GTP-binding proteins, are a family of enzymes thatserve as molecular switches in regulating
a number of signal transduction pathways
including cellular growth, apoptosis and differentiation. These proteins cycle between
an inactive, GDP-bound state and an active,
GTP-bound state. Activated GTPases exert
their effects by activating a variety ofdownstream effector proteins such as Raf
and PI3K. Activation of effector proteins inturn initiates phosphorylation cascades thatmodulate a number of different processes
in the cell (Figure 2).
Activating
signalPi

Ras-GDP

H2O

Ras-GTP

+

GTP
GDP

Effectors
Cellular responses
Figure 2: The Ras activation pathway.
Ras GTPase cycles between its inactive, GDP-bound form and its
activated, GTP-bound form.

Aberrant Ras signaling causes many cancers
Ras GTPase is of particular interest because
it is involved in many different pathways,
and because aberrant regulation by Ras has
been implicated in a number of diseasestates. Normally, GTPase-signaling cascades
are only transiently activated because theintrinsic hydrolyzing activity of GTPases
gradually converts GTP to GDP, leading toinactivation. However, a number of mutations in the ras gene have been identified
that cause Ras to remain constitutively in
its active, GTP-bound form. This results incontinuous stimulation of cellular proliferation. Consequently, constitutively active,
mutant forms of Ras GTPase are estimatedto be present in ~30% of all human cancers.

45 min45 min45 minAdd developing
and stop solutions,then quantify

Add cell
extract toplate

Cell extractcontaining activatedRas GTPase

GST-Raf-RBD
coated plate

Figure 3: Flowchart of the Ras GTPase Chemi Kit.
Cell extract is added to a glutathione-coated plate that contains immobilized GST-Raf-RBD protein. Activated Ras in the extract binds to the Raf-RBD protein.
Addition of primary & secondary antibodies and developing solution followed by reading on a luminometer enables sensitive quantification of activated Ras.

Ras GTPase Chemi ELISA advantages• More sensitive – assay uses only 25 µgof extract, which is 20-fold less thanpull-down/Western methods• Better results – quantitative readouts
make it easier to compare results• Less effort – no need to run gels ordevelop Western blots• Save time – results in less than 5 hours• Versatile – assay activated extracts
from cells or tissue samples, or study
recombinant Ras protein
The Ras GTPase Chemi ELISA method
Because activated Ras binds specificallyto the Ras-binding domain (RBD) of Raf
effector protein, Raf-RBD can be used asa probe to isolate Ras-GTP. Active Motif’s
new Ras GTPase Chemi ELISA Kit contains
a Raf-RBD protein that is fused to GST anda 96-well, glutathione-coated assay plate.
GST-Raf-RBD is first incubated on the platefor one hour to immobilize the captureprobe. Addition of sample to the plate results in the binding of activated Ras to the
Product
Ras GTPase Chemi ELISA Kit

6

Add anti-IgG
HRP conjugate

Add primary
antibody

Raf-RBD. A primary antibody specific for
Ras is then added to the individual wells,followed by an HRP-conjugated secondary
antibody and developing reagent (Figure 3).
The plate is then read on a luminometer,
which provides a sensitive and quantitative
chemiluminescent readout of activated Ras(Figure 1, page 1).
Try the quantitative, sensitive assay
The Ras GTPase Chemi ELISA Kit makes
it fast and easy to detect and quantify
activated Ras GTPase. The kit is ideal forthe study of novel signaling pathways that
activate Ras, as well as for determining if aparticular malignancy is related to inapproriately activated, oncogenic Ras. Additional
assays to detect other GTPase proteins
are in development. Please give us a call,return the enclosed reply card or log on towww.activemotif.com/gtpase for complete
information, downloadable manuals andto find out about new additions to thisinnovative product line. To get to the best
Ras activation assay available for Ras, order
the Ras GTPase Chemi ELISA Kit.
FormatCatalog No.

1 x 96-well plate

52097

Toll Free — 1 877 222 9543

APRIL 2005 • VOLUME 6 • NUMBER 1

Nuclear Receptor Assays

A Complete Solution for Studying Nuclear Receptor Protein Activity

Monitor ligand activation with NR Peptide
Studying the agonist/antagonist effects
of potential drug targets is an importantelement of nuclear receptor-targeted drug
discovery. Active Motif’s NR Peptide ELISAs
are specifically designed to capture ligandactivated nuclear receptor and can be usedwith both cell extracts and proteins. EachNR Peptide ELISA Kit provides a 96-wellplate that is coated with a Capture Peptidethat includes the consensus-binding motifof the nuclear receptor’s co-activator.
Addition of sample results in binding ofligand-activated nuclear receptor to the
Capture Peptide. Each well is then incubated with a primary antibody specific forthe nuclear receptor of interest, followedby an HRP-conjugated secondary antibody
and developing solution to provide aneasily quantified readout. This enables you
to quickly and quantitatively measure theagonist/antagonist effects of target compounds on the binding of ligand-activatednuclear receptors (Figure 1).

Assess DNA-binding activity of NRs
Inappropriate nuclear receptor signaling is associated with numerous diseases
including cancer, asthma and arthritis,which makes NRs promising drug targets.
Because the end point of nuclear receptor activation is DNA binding, monitoringchanges in the DNA-binding activity ofa target nuclear receptor can serve as an
ideal biomarker. Classical methods such asradioactive gelshifts and time-consuming
reporter gene assays are not well suited tothis application. Active Motif’s TransAM™
Kits provide an innovative alternative tothese classical assays. TransAM Kits use acombination of DNA binding and antibody
detection to give a specific, quantitative
readout of DNA-binding activity from all
sample types. (See page 1 for more details.)
Quantify total NR with Sandwich ELISAs
In order to fully examine the activation ofa given nuclear receptor, it is important
to be able to quantify the total levels of agiven nuclear receptor within a sample. The
new NR Sandwich ELISAs offer a simple,
rapid method to quantify the total amountof nuclear receptor protein present inboth cell and tissue samples. NR Sandwich
Kits utilize the Sandwich ELISA-based
method that is an improvement over othermethods used to study proteins, suchas Western blotting. Using NR Sandwichmeans that there is no need for gels,blotting or long, tedious incubations. The
Product

Figure 1: ERα agonism/antagonism dose-response curves.
Nuclear extracts from the breast cancer cell line MCF-7 are incubatedin wells of the NR Peptide ELISA ER plate in the presence of 100-foldserial dilutions (from 10-6 to 10-14 M) of the agonist compounds
diethylstilbestrol (DES) and estradiol, and the antagonist compounds
tamoxifen and clomiphene. Only ligand-activated ER protein can bindto the Capture Peptide immobilized in the plate. Bound ER is specifically detected with ERα antibody and quantified using a secondary
antibody and Detection Solution.

www.activemotif.com

96-well format 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 make studying nuclear receptor proteins both fasterand more accurate than using traditional
methods. Whether you are interested in
DNA-binding activity, activation state,
protein level or agonist/antagonist effects,
Active Motif has the product you need.

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 using NR Sandwich AR.
Different amounts of nuclear extracts from three human prostatecancer cell lines, LNCaP, PC-3 and DU 145, were analyzed for levels of
AR protein using the NR Sandwich AR Kit.

A collection of NR abs & proteins
Active Motif’s extensive line of antibodies provide superior performance andreliable results. We offer over 200 highly
characterized antibodies directed against
transcription factors and nuclear receptors,
including antibodies for GR, PPARα and γ,
PXR, RAR-β, -β2 and -γ, RXR-α and -β,
and VDR. In addition, Active Motif offers anumber of recombinant nuclear receptor
proteins that are ideal for use as positive
controls, in in vitro screening studies and inmany other applications.
Get the tools you need for NR research
To get complete information on all ofActive Motif’s tools for studying nuclear
receptors, please give us a call, visit our
website or send in the enclosed reply card.
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

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

7

Antibodies

New Antibodies for Chromatin and DNA Methylation
Active Motif has recently added a numberof antibodies related to epigenetics andsignal transduction to its already extensive

line of antibodies. The tables below list
these recent additions. Active Motif alsooffers numerous cell extracts that are

ideal positive controls for use with its kits
and antibodies. For complete information,please visit www.activemotif.com.

FormatApplication

React.

Cat. No.

Product

FormatApplication

React.

Cat. No.

APE pAb100 µg

WB

H

39200

MBD1 mAb

100 µg

WB

H

39215

APEXL2 pAb

200 µl

WB

H

39201

MBD3 mAb

100 µg

WB

H

39216

INHAT-1/TAF-1α/TAF-1β pAb

200 µl

WB

H

39202

MBD4 pAb

100 µg

WB

H

39217

CGBP pAb

200 µl

WB

H

39203

MeCP2 pAb

100 µg

WB

H

39218

DNMT1 mAb

100 µg

Ch, IHC, IP, WB

H, M

39204

MLH-1 mAb

100 µg

WB

H

39219

DNMT2 pAb

100 µg

WB

H, M

39205

MRE11 pAb

100 µg

WB

H

39220

DNMT3A mAb

100 µg

Ch, IF, IHC, WB

H, M

39206

PMS2 mAb

100 µg

WB

H, M

39221

DNMT3B mAb

100 µg

Ch, IF, IP, WB

H, M

39207

Rad17 (Phosphorylated) pAb

100 µgIF, WBH

39222

HDAC11 pAb

200 µl

WB

H, M

39208

TRF2 pAb

100 µg

Ch, IP, WB

H, M

39223

Histone H2A pAb

100 µg

WB

H

39209

UBE2N pAb

100 µgIP, WBH

39224

Histone H2B pAb

100 µg

WB

H

39210

UBE2V1a pAb

100 µg

WB

H

39225

Histone H3 (Phosphorylated) mAb

100 µg

WB

H

39211

UBE2V2 pAb

100 µg

WB

H

39226

Histone H4 pAb

100 µg

WB

H

39212

INHAT-2/pp32 pAb

200 µl

WB

H

39213

LIG1 pAb

200 µl

WB

H

39214

CONTACT INFO

Product

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: sales@activemotif.com

EUROPE
104 Avenue Franklin Roosevelt–Box 25
B-1330 Rixensart, Belgium
Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
United Kingdom Free Phone: 0800 169 31 47
Other countries, Direct: +32 (0)2 653 0001
Fax: +32 (0)2 653 0050
Email: eurotech@activemotif.com

JAPAN
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
Email: japantech@activemotif.com

8

Application Key:
Ch = Chromatin Immunoprecipitation; IF = Immunofluorescence; IHC = Immunohistochemistry;
IP = Immunoprecipitation; WB = Western blot
Reactivity (React.) Key:H = Human; M = MousePRSRT STD
US POSTAGEPAID
PERMIT 236
92054