IN THIS ISSUE

Epigenetics, Chromatin Biology & Cell Signaling
2 NEW: RNA ChIP-IT™ for Studying
Chromatin-associated RNAs

7 Reproducible Bisulﬁte Conversion for
Accurate Analysis of DNA Methylation

2 FREE: New Histone Modiﬁcations Poster

7 Simple Screen for DNMT Activity or Inhibition

3 NEW: Simple Formaldehyde Detection ofHistone Demethylase Activity

8 Get Pure Fractions with Every
Sample Preparation

3 NEW: Pre-made HeLa Mononucleosomes
Simplify Substrate Analysis

8 Non-radioactive Evaluation ofProtein:DNA Binding

4 NEW: MODiﬁed™ Array Labeling Kit andFree Software for Analysis

9 Signaling Pathway Readouts by In-Cell ELISA:
Simple, Quick, and Scalable

5 Explore Chromatin Biology with
Recombinant Histone Modifying Enzymes

10 SUMOlink™ Enables Efﬁcient Analysis of theEffects of SUMOylation

5 Rigorously Tested Antibodies to
Chromatin Proteins

10 Sensitive Reporter Assay for Better Results

6 NEW: Study Both Types of DNA Methylation
with the Proper Controls

11 Chariot™ Delivers Functional Protein
Directly into Living Cells

11 Extremely Affordable Competent E. coli

Chromatin IP of RNA & Free Poster Offer

NEW: RNA ChIP-IT™ for Studying Chromatin-associated RNAs
Evidence is building that RNA-directed processes play a critical role in orchestrating chromatin architecture andepigenetic memory. Nucleic acids puriﬁed from chromatin are 2-5% RNA; these RNAs are non-coding sequences thatplay important roles in chromatin structure and transcriptional silencing. But, characterizing these RNAs by ChIP
techniques is difﬁcult due to the complexity of chromatin and the high amounts of DNA in chromatin.

The RNA ChIP-IT method
RNA ChIP-IT uses a modiﬁed ChIP protocol that has been optimized for RNA
preservation and recovery. RNA-protein
interactions are ﬁxed with formaldehyde, and chromatin shearing is combined with DNase treatment to yield
RNA/protein complexes that can be
immunoprecipitated with antibodies tospeciﬁc proteins. Cross-links are subsequently reversed; RNA is recovered andagain treated with DNase to ensure theabsence of DNA. The optimized method
is quick and has been successfully used
to study several non-coding RNAs in thechromatin context.
Advantages of the RNA ChIP-IT Kit• Speciﬁcally tailored to study
chromatin-associated RNA• Designed to remove DNA while
maintaining RNA integrity

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_dfkjh[Yel[hoOptimized RNA-ChIP
To make the characterization of the roleof RNA in genome regulation possible,
Active Motif has leveraged its expertisein ChIP to develop the ﬁrst of its kind
kit for RNA-ChIP. The RNA ChIP-IT™ Kit
was designed to study RNA-protein
interactions in a chromatin context, andoptimized to recover RNA for RT-PCR
analysis. It contains sufﬁcient materialfor 25 assays and uses protocols thatutilize magnetic beads, which improve
results while reducing time and effort.

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Figures 1 & 2: Real-time RT-PCR analysis and % input recoveries of Suz12/normal rabbit IgG RNA-ChIP samples.
The RNA ChIP-IT Kit was used on 10 µg samples of DNase I-treated HeLa chromatin with 10 µl of Suz12 antibody (Catalog
No. 39357) and 2 µg of Normal rabbit IgG. Real-time RT-PCR was performed using primers for the lincRNA SFPQ locus. The
amplification plot (left) and % input recoveries (right) are shown. Dividing the input recovery of the Suz12 antibody by thatof the rabbit IgG indicates a 141-fold enrichment of the lincSFPQ region with the Suz12 antibody.

• Step-by-step protocols for ﬁxation of chromatin, sonication andimmunoprecipitation, all optimized
for RNA preservation• Includes all RNase and proteaseinhibitors at precise concentrations• Separate control kit available with
control antibody and primers
In contrast to other kits designed to
study RNA-protein interactions, called
RIP kits, RNA ChIP-IT is designed speciﬁcally to extract and immunoprecipitate
RNA from chromatin, and to solve theassociated challenges in extracting chromatin while preserving RNA integrity,
and removing all DNA, for a clean resultthat is attributable to RNA alone. The
RNA ChIP-IT Kit is the solution optimized for the Epigeneticist studying
RNA, rather than the RNA biologist.
FormatCatalog No.

RNA ChIP-IT™

25 rxns

53024

RNA ChIP-IT™ Control Kit – Human

5 rxns53025

North America 877 222 9543

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Europe +32 (0)2 653 0001

Take control of your RNA-ChIP
Also available is the RNA ChIP-IT Control
Kit, which was designed to be used withRNA ChIP-IT. The kit contains positive &negative control antibodies and RT-PCR
primers for the lincSFPQ locus. These areused to verify that the chromatin sample
was prepared correctly, and to validate
antibodies for use in RNA-ChIP.
Start investigating the role of RNA today
For more complete information, including a downloadable product manual, visit
www.activemotif.com/rnachip.

Get Active Motif’s New
Histone Modiﬁcations Poster
Request your free copy of our
new poster, which details
histone modiﬁcations and theirreaders, writers and erasers, at:www.activemotif.com/poster.

Japan +81 (0)3 5225 3638

www.activemotif.com

October 2010 • volume 11 • number 3

Histone Demethylation & Histone Substrates

NEW: Simple Formaldehyde Detection of Histone Demethylase Activity
The ﬂuorescent Histone Demethylase Assay is a simple assay to analyze the efﬁciency of lysine speciﬁc demethylase
enzyme (LSD1, also known as KDM1) samples, or to screen compounds for changes in histone demethylation activity.
The Histone Demethylase Assay is designed to detect the formaldehyde released from the reaction of LSD1 with amethylated substrate. As the LSD1 enzyme demethylates the recombinant histone H3K4me2 substrate, formaldehyde
is released as a by-product, which then reacts with the Detection Reagent to generate a ﬂuorescent signal equivalent
to the overall production of formaldehyde.
Histone Demethylase Assay advantages• The recombinant histone H3K4me2 substrate used in theassay mimics a native histone substrate, providing you withresults that more closely resemble in vivo conditions• Complete assay includes a Demethylation Standard forformaldehyde quantiﬁcation and an LSD1 enzyme as apositive control protein<ehcWbZ[^oZ[Yedl[hi_ed[\\_Y_[dYo

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Product

FormatCatalog No.

Histone Demethylase Assay (Fluorescent)

48 rxns

53200

Recombinant LSD1 protein, active

50 µg

31334

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• Simple ﬂuorescent assay detects the formaldehyde by-productusing an excitation wavelength of 410 nm and an emission
wavelength of 480 nm

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CJ;("I\ZfdY`eXek?*B+d\)gifk\`eFigure 1: Comparison of different histone substrates
and their effect on LSD1 demethylase efﬁciency.
The positive control LSD1 enzyme from the Histone
Demethylase Assay was used to evaluate demethylase
activity using either a peptide or the kit’s recombinanthistone protein. One µg of LSD1 was tested with either
70 µM H3K4me2 peptide or with 13 µM recombinanthistone H3K4me2 protein. LSD1 was able to convert 73%
of the recombinant histone substrate into a formaldehyde by-product, yet it was only able to convert 14% of
the peptide substrate into a formaldehyde by-product.

NEW: Pre-made HeLa Mononucleosomes
Simplify Substrate Analysis

New RecombinantHistones Available

Histone analysis can be complicated when working with nucleosomal arrays,
due to the number of potential binding interactions that are possible on
the histone tails, histone globular domain and the nucleosomal DNA. Active
Motif’s new pre-made HeLa Mononucleosomes reduce the complexity ofpotential interactions, enabling simpliﬁed substrate analysis with histone
methyltransferases, histone demethylases, histone acetyltransferases andhistone deacetylases. To view a list of Active Motif’s histone modifying
enzymes, please visit www.activemotif.com/hismodenz.

Active Motif is the ﬁrst company
to offer recombinant histones withacetylation and site-speciﬁc mono-,
di- and trimethylation. These recombinant histones can be used as controls
for histone antibodies, substrates forhistone modiﬁcation enzymes, or to
generate chromatin in vitro using
Active Motif’s Chromatin Assembly Kit(Catalog No. 53500).

Product

FormatCatalog No.

HeLa Mononucleosomes

10 µg

53300

www.activemotif.com

Japan +81 (0)3 5225 3638

Europe +32 (0)2 653 0001

For an up-to-date list of our more than
20 recombinant histones, please visit
www.activemotif.com/recombhis.

North America 877 222 9543

3

Modified Histone Peptide Array

NEW: MODiﬁed™ Array Labeling Kit and Free Software for Analysis
The MODiﬁed™ Histone Peptide Array* is a useful research tool that can be used to screen antibodies, proteins andenzymes for interactions with histones and their post-translational modiﬁcations. Each array contains 384 different
histone modiﬁcation combinations in duplicate. Modiﬁcations include acetylation, methylation, phosphorylation andcitrullination on the N-terminal tails of histones H2A, H2B, H3 and H4. Active Motif’s new MODiﬁed™ Array Labeling
Kit contains all the necessary buffers and reagents for ECL-based detection of the MODiﬁed Histone Peptide Arrays.
How do the MODiﬁed Arrays work?
The MODiﬁed Histone Peptide arrays
contain 384 unique histone peptides
spotted in duplicate. Each 19mer peptide
contains up to four separate modiﬁcations to allow researchers to study not
only individual sites, but also to determine if neighboring modiﬁcations altersite recognition and binding.
The simple array protocol works likea Western blot, using either ECL or
colorimetric detection systems. The image is then captured using ﬁlm or a CCD
camera; no special equipment is needed(Figure 1). For analysis, Active Motif’s free
Array Analyse Software can be used.
The MODiﬁed Histone Peptide Arrays
are available individually, or in packs ofﬁve. For a complete solution, Active
Motif’s new MODiﬁed Array Labeling
Kit provides all the reagents needed for
ECL-based detection.
What’s in the box?
The MODiﬁed Array Labeling Kit contains blocking buffer, wash buffer and
ECL reagents for reliable and reproducible chemiluminescent detection. For
added convenience, an antibody torecognize the arrays’ control c-Myc
peptide and HRP-conjugated secondary
antibodies are also included.
* CelluSpots™ arrays are manufactured underlicense by INTAVIS Bioanalytical Instruments AG
and sold through Active Motif as MODiﬁed™
Histone peptide Arrays.

4

North America 877 222 9543

Free software for analysis
Active Motif’s Array Analyse Software
is a free program designed for use with
the MODiﬁed Histone Peptide Arrays.
This PC-compatible software will analyze
the spot intensities from the MODiﬁed
Array and generate a graphical analysisof the histone modiﬁcation interactions.
Information about spot intensity, averages and errors can be saved in Excelcompatible ﬁles. For added convenience,
up to three individual modiﬁcations
can be displayed in superposition to
the experimental data, enabling better
visualization of neighboring effects.
MODiﬁed Array advantages• Histone speciﬁc – unique arraypanel tests for speciﬁc histone
modiﬁcations• Study neighboring effects –each peptide contains up to
four modiﬁcation combinations,enabling analysis of the effects
of neighboring modiﬁcations• Detects like a Western blot – fastand easy to use; works with either
ECL or colorimetric detection• Free analysis software – the Array
Analyse Software program enables
easy analysis of spot intensity andmodiﬁcation interactions

7

8

9

Figure 1: Images of ECL detection of MODiﬁed Histone
Peptide Arrays treated with HMT G9a.
MODified histone peptide Arrays were treated withA) 25 µM G9a methyltransferase (Catalog No. 31327),
B) 25 µM G9a mutant H904K (Catalog No. 31328), C)
no enzyme control, overnight in the presence of 1 mM
AdoMet. The arrays were detected using a Histone H3
dimethyl Lys9 antibody. Novel methylation sites wereobserved on array A, which was treated with wild-type
G9a histone methyltransferase, showing the activity of
the histone modifying enzyme on the peptide substrate.

Visit www.activemotif.com/modiﬁed
to view a complete list of the peptides
available on the MODiﬁed™ Histone
Peptide Array, to learn more about theMODiﬁed™ Array Labeling Kit, or to
download the free Array Analyse
Software and instruction manual.

Product

FormatCatalog No.

MODiﬁed™ Histone Peptide Array

1 array
5 arrays

13001
13005

MODiﬁed™ Array Labeling Kit

5 rxns13006

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

October 2010 • volume 11 • number 3

Histone Modifying Enzymes & Chromatin Abs

Explore Chromatin Biology with Recombinant Histone Modifying Enzymes>CJWi[7Yj_l_jo

The eukaryotic genome is packaged into the nucleus through the compaction afforded by the incorporation of DNA
into chromatin. The primary structural components of chromatin are the highly conserved histone proteins, aroundwhich DNA is wrapped and organized. Histones are subject to a variety of reversible post-translational modiﬁca-&&&tions that are tightly regulated; these include phosphorylation,,&&&acetylation, methylation and ubiquitylation. These modiﬁcations
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represent important regulatory events that govern the accessibil)&&&ity and function of the genome.
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and removed by enzymes that are generally part of large multiCBBª]
subunit protein complexes recruited to chromatin by sequence- Figure 1: Recombinant MLL activity measured using a ﬂuorescent histone H3lysine 4 methyltransferase assay.
speciﬁc DNA binding proteins. These histone modifying enzymes Increasing amounts of MLL protein (Catalog No. 31338) were incubated with thesubstrate and then the reaction was developed. MLL activity is measured inare important regulators of genome function, and studying their relative fluorescence units.
function offers insight into the mechanisms that regulate processes dependent upon the genome.
Active Motif offers a variety of recombinant histone modifying enzymes, such as Acetyltransferases, Deacetylases,
Demethylases & Methyltransferases, for use in your exploration of chromatin biology. To see our complete offering,please visit www.activemotif.com/hismodenz. To get them at 15% off, please visit www.activemotif.com/promo.

Rigorously Tested Antibodies to Chromatin Proteins
The proteins associated with chromatin are important to the organization of genomic DNA and the regulation of its
activity and accessibility. Active Motif is committed to providing high-quality reagents for the study of epigenetics
and chromatin biology. We specialize in developing antibodies to study
histones, chromatin proteins and transcription factors, which are thenvalidated for use in the applications you need them for: Western blot,
chromatin immunoprecipitation (ChIP) and immunoﬂuorescence.
Chromatin Protein Antibody Categories• Non-histone chromatin proteins
Figure 1: HDAC2 detected by immunoﬂuorescence.
HeLa cells were stained with HDAC2 monoclonal
antibody (Clone 3F3, Catalog No. 39533) at a dilutionof 1:1,000. Red: HDAC2 staining. Green: alpha-Tubulin
mouse monoclonal (Clone 5-B-1-2, Catalog No. 39527)
conjugated to Chromeo™ 488.

• Histone modifying enzymes• Polycomb group proteins• Chromatin remodeling proteins

To see our complete offering of antibodies to chromatin-associated proteins, visit www.activemotif.com/chromabs.
For our complete list of antibodies to histone and histone modiﬁcations, visit www.activemotif.com/histoneabs.
To ﬁnd out more about our antibody development and validation process, visit www.activemotif.com/abdevel.
www.activemotif.com

Japan +81 (0)3 5225 3638

Europe +32 (0)2 653 0001

North America 877 222 9543

5

Methylated DNA Standards

NEW: Study Both Types of DNA Methylation with the Proper Controls
Methylation of DNA at the 5´ position of cytosines is an important regulator of cellular function, inﬂuencing gene
expression, cellular identity and disease development. With the recent discovery of the importance of a novel formof DNA methylation, 5-hydroxycytosine methylation, it is crucial to have the right tools for your experiments. No
matter which type of DNA methylation you are studying, Active Motif’s new Methylated DNA Standard Kit can help.
The importance of DNA methylation
In mammals, DNA is methylated at the
5´ position of cytosines and is foundprimarily at clusters of CpG residues that
reside in the promoter region of half ofall human genes. Non-CpG methylation,
however, is also very abundant in certain
cell types such as stem cells. DNA
methylation is involved in many cellularfunctions including embryonic development, genetic imprinting, X chromosome
inactivation and control of gene expression. DNA methylation and chromatin
modiﬁcations interact to bring abouttranscriptional silencing. Aberrant DNA
methylation patterns are also associatedwith certain cancers as well as developmental abnormalities.
A novel type of DNA methylation, 5-hmC
In addition to cytosine methylation,
several papers have been published recently describing the relative abundanceof 5-hydroxymethylcytosine (5-hmC)
in speciﬁc cell types. 5-methylcytosine(5-mC) is converted into 5-hmC by the
TET family of iron-dependant oxygenases. 5-hmC can be replaced by cytosine

Figure 1: Agarose gel analysis of samples supplied in
the Methylated DNA Standard Kit.
Five hundred ng of each DNA standard was loaded on a
2.5% agarose gel.
Lane 1: unmethylated DNA.
Lane 2: 5-methylcytosine containing DNA.
Lane 3: 5-hydroxymethylcytosine containing DNA.

6

North America 877 222 9543

study of either 5-methylcytosine or
5-hydroxymethylcytosine methylation. It
has been validated for use in MeDIP and
dot blot and may be useful in gel shifttype experiments studying proteins thatbind 5-mC or 5-hmC methylated DNA.

Figure 2: Dot blot analysis of Methylated DNA
Standard Kit samples.
DNA (50 ng) from the three different samples was spotted onto a positively charged nylon membrane.
Top panel: Dot blot probed with 5-Methylcytidine
antibody (Clone 33D3) (Cat. No. 39649, 1:1,000 dilution).
Bottom panel: Dot blot probed with 5-Hydroxymethylcytidine antibody (Cat. No. 39769, 1:5,000 dilution). Lane 1:
Single-stranded DNA. Lane 2: Double-stranded DNA.

by DNA repair proteins, thus conversionof 5-mC into 5-hmC may represent apathway by which DNA is demethylated.
Active Motif’s Methylated DNA Standard
Kit includes three recombinant DNA
standards derived from the APC gene
regulatory region: unmethylated DNA,
5-methylcytosine methylated DNA and
5-hydroxymethylcytosine methylatedDNA. This kit (which also includes PCRprimers speciﬁc to the APC gene) canbe used to provide controls for experiments studying the different types of
DNA methylation. It can be used in the

Methylated DNA Standard advantages• Your experiments can now utilize
controls for both forms of DNA
methylation – 5-methylcytosine
and 5-hydroxymethylcytosine• Includes primers that work in
endpoint and real-time PCR• Validated for use in MeDIP and
dot blot experiments• DNA is methylated at both CpG
and non-CpG sites
Full line of DNA Methylation products
Due to the importance of DNA methylation in development and disease, muchof today’s epigenetic research depends
on the ability to accurately detect andquantify DNA methylation. Active Motif
offers a broad range of products for
DNA methylation analysis, from methylation enrichment kits to recombinant
proteins and antibodies against proteins
involved in DNA methylation. For a complete list of products available for the
study of DNA methylation, please visit
www.activemotif.com/dnamt.

Product

FormatCatalog No.

Methylated DNA Standard Kit

3 x 2.5 µg

55008

5-Hydroxymethylcytidine antibody (rabbit IgG)

100 µg39791

5-Hydroxymethylcytidine antibody (rabbit serum)

100 µl39679

5-Methylcytidine antibody (mouse IgG)

50 µg

39649

Fully Methylated Jurkat DNA

10 µg

55003

Jurkat genomic DNA

10 µg

55007

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

October 2010 • volume 11 • number 3

Bisulfite Conversion & DNMT Assays

Reproducible Bisulﬁte Conversion for Accurate Analysis of DNA Methylation
Bisulﬁte conversion is a useful tool to obtain single nucleotide resolution information about the methylation statusof a particular region of DNA. The MethylDetector™ Bisulﬁte Modiﬁcation Kit makes DNA methylation analysis fastand efﬁcient by providing optimized reagents, time-saving DNA puriﬁcation columns and positive control PCR
primers for assay validation.
Proven controls verify your success
DNA methylation analysis often uses
bisulﬁte to convert unmethylated
cytosines to uracils, leaving methylated
cytosines unchanged. The DNA is thenPCR ampliﬁed and analyzed by sequencing or restriction digest, which can be
costly and time-consuming. Thus, conﬁrming that the conversion was successful before analyzing the samples is a big
beneﬁt. To that end, the MethylDetector
Kit provides positive control PCR primers
speciﬁc for bisulﬁte-converted DNA, so
you can conﬁrm the conversion worked
before starting the analysis (Figure 1).
For additional information aboutbisulﬁte conversion, or to see a complete list of Active Motif’s DNA methylation antibodies and assay kits, please
visit www.activemotif.com/dnamt.

M“MethylDetector’s reliable andconsistent results improved myanalysis of imprinted genes, andworked well in combination with
the Sequenom EpiTYPER system.”– Benedetta Izzi, Katholieke
Universiteit Leuven, Belgium
Purchase a MethylDetector™ Kit by
November 30th and save 10%. Simply citecode AUP2 at the time of the order. For
complete promotional details, please
visit www.activemotif.com/promo.

1

2

3

4

5

500 bp400 bp300 bp200 bp100 bpFigure 1: Reproducible conversion by MethylDetector.
MethylDetector was used for bisulfite conversion of
3 different DNA samples (Lanes: 1-3) and a control with
no DNA (Lane 4). PCR was performed on these samples
and an unconverted DNA control (Lane 5) using the kit’s
control PCR primers. The presence of PCR product inonly the converted samples demonstrates the efficiency
and reproducibility of the MethylDetector Kit.

Product

FormatCatalog No.

MethylDetector™

50 rxns

55001

Simple Screen for DNMT Activity or Inhibition

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Active Motif’s DNMT Activity / Inhibition Assay is a fast, user-friendly assay
to speciﬁcally detect DNA methyltransferase (DNMT) activity from puriﬁedproteins or nuclear extract samples, without the need for radioisotopes.

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Unique method enhances sensitivity
The DNMT Activity / Inhibition Assay is
unique in that it utilizes a methyl CpG
binding domain protein (MBD) to detect
methyltransferase activity. MBD proteinsare capable of binding methylated DNA
with a higher afﬁnity than antibody approaches, which increases the sensitivityof the assay. With this method, as little
as 0.5 ng of puriﬁed enzyme or 0. 5 µg of
nuclear extract can be detected.
Learn more at www.activemotif.com/dnmt.

www.activemotif.com

DNMT Assay advantages• Non-radioactive – colorimetricassay is easily quantiﬁed at 450 nm• Sensitive – unique MBD protein
approach enhances the sensitivityof detection (Figure 1)• Fast – assay can be completed inless than 3 hours

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Figure 1: Puriﬁed DNMT1 activity.
The DNMT Activity / Inhibition Assay was used togenerate a standard curve from 0.313 - 10 ng per well of
the included DNMT1 control enzyme. The assay was able
to detect DNMT1 activity from as little as 0.313 ng of
DNMT1 with a 1.5 hour incubation time and a 3 minutedeveloping time.

Product

FormatCatalog No.

DNMT Activity / Inhibition Assay

1 x 96 rxns

55006

Recombinant DNMT1 protein, active

10 µg

31335

Japan +81 (0)3 5225 3638

Europe +32 (0)2 653 0001('

Fkh_\_[Z:DCJ'd]%m[bb

North America 877 222 9543

7

Sample Preparation & EMSA

Get Pure Fractions with Every Sample Preparation
To obtain results you can rely on, it is important to know that your starting material is pure. With Active Motif’s
Nuclear Extract Kit and Mitochondrial Fractionation Kit, it is possible to have conﬁdence in your sample preparationand know that your extract is not contaminated with proteins from other cellular compartments (Figure 1).
The Nuclear Extract Kit is ideal for the
preparation of nuclear, cytoplasmic
and whole-cell extracts from mammalian cells and tissues. The included
phosphatase and protease inhibitors
help to preserve protein modiﬁcations.
Active Motif’s unique Mitochondrial
Fractionation Kit isolates highly enriched, segregated mitochondrial andcytosolic fractions from mammalian celllines using a simple procedure that does
not require ultracentrifugation or theuse of toxic chemicals.
Get 10% off the purchase of any Nuclear
Extract or Mitochondrial Fractionation
Kit between now and November 30th.
Simply cite code AUP1 at the time of theorder. For complete details, please visit
www.activemotif.com/promo.

Figure 1: Speciﬁc extraction of nuclear and cytoplasmic extracts.
Nuclear and cytoplasmic extracts were isolated using the Nuclear Extract Kit. NFκB activation was then assayed with theTransAM™ NFκB p50 Kit using increasing amounts of each extract. This data demonstrates the kit’s high specificity; because
activated NFκB translocates into the nucleus, only nuclear extract from stimulated cells should contain activated NFκB.

Product

FormatCatalog No.

Nuclear Extract Kit

100 rxns
400 rxns

40010
40410

Mitochondrial Fractionation Kit

100 rxns

40015

Non-radioactive Evaluation of Protein:DNA Binding
Looking for an easier way to assess protein:DNA interactions? Active Motif’s
Gelshift™ Chemiluminescent Kit is a simple, non-radioactive electrophoretic
mobility shift assay (EMSA) that can be used to screen for protein binding
to DNA. Simply incubate cell extracts or puriﬁed factors with a biotin endlabeled probe containing the consensus binding site of interest; then resolvethese reactions on a native polyacrylamide gel. Samples in which the protein of interest bound the target DNA will migrate slower than DNA alone
resulting in a “shift” of the labeled DNA band. The non-radioactive format
does not sacriﬁce sensitivity when compared to 32P or digoxigenin-labeled
methods (Figure 1).
Product

FormatCatalog No.

Gelshift™ Chemiluminescent EMSA

100 rxns

37341

8

North America 877 222 9543

Europe +32 (0)2 653 0001

Gelshift™
Chemi EMSA

32
PLabeledDigoxigenin
Labeled

Exposure Time

3 minutes

16 hours

60 minutes

HeLa Extract
Competitor

- + +
- - +

- + +
- - +

- + +
- - +

Figure 1: Gelshift EMSA gives better results.
Comparison of Gelshift Chemiluminescent EMSA toradioactive and digoxigenin-based methods showthat detection of a 22 bp duplex probe specific for
Oct-1 with HeLa nuclear extract using the Gelshift
Chemiluminescent EMSA gives better results in less time.

Japan +81 (0)3 5225 3638

www.activemotif.com

October 2010 • volume 11 • number 3

Phospho-Protein Detection

Signaling Pathway Readouts by In-Cell ELISA: Simple, Quick, and Scalable
Active Motif’s FACE™ Kits provide a simple, sensitive, cell-based method formonitoring the status of signaling pathways by In-Cell ELISA.
2

3

4

OD450 nm1

Is AKT active in my cell system? Howabout ERK, or p38? Does this have aneffect on what is seen in the nucleus?
Study signaling pathways with FACE Kits.
The regulation of chromatin and otheraspects of the nucleus is greatly dependent on the context of signaling pathways active in a particular cell type. As
you study the nucleus, take some time
to look at the signaling pathways active
in your cell type, and make new associations between signaling and the nucleus.
Rather than the laborious process ofmaking extracts, running gels, and Western blots, why not try our Fast Activatedin-Cell ELISA (FACE) Kits? We’ve done all
the validation of antibodies and protocols, ensuring that only the cognate
protein is recognized. You only need to
grow your cells (in a 96-well dish), treat
as appropriate, ﬁx, stain and detect. In
this way you can learn important aspectsof cell regulation with a minimum of effort, to dovetail with discoveries about
the status of the nucleus in those cells.
In-cell ELISAs: Cell Signaling made easy
Active Motif’s In-cell ELISAs can be completed with under 3 hours of hands-on
time. Cells are grown in 96-well culture
plates, stimulated, ﬁxed with formaldehyde, and probed in parallel with aphospho-state independent antibody tothe protein of interest, and a phosphospeciﬁc antibody to a regulatory siteon the same protein. Detection iscolorimetric or luminescent; we provide
both formats. When you compare thetwo signals, you see total protein versus
phospho-protein, readily giving you the

43 kDa1

2

3

4
43 kDa

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

A

B

Phospho p38
Total p38

C

0

0.1
1
10
Anisomycin induction (µg/ml)

Figure 1: Phospho and total p38 MAPK assays.
Macrophage 4/4 cells were grown in 10 cm dishes to
80% confluency, serum-starved for 16 hours and stimulated with anisomycin for 15 minutes. Cell lysates weremade and Western blots performed using phospho- (A)and total-p38 antibodies (B). For FACE, 4/4 cells weregrown in 96-well plates, stimulated as above, fixed andthen assayed in triplicate using the FACE p38 Kit (C). Data
were corrected for cell number through use of the kit’s
Crystal Violet Dye. Western blot data provided courtesyof Dr. Henri H. Versteeg and Dr. Maikel P. Peppelenbosch.

Add anti-IgG
HRP conjugate

Add primary
antibody
3 hrs.

1 hr.

Cells arefixed following
stimulation

P

P

P

Add developing
and stop solutionwww.activemotif.com

Cells cultured in
96-well plate

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FACE advantages• Quick – less than 3 hours ofhands-on time
• High Information Yield – works
on 96-well plates• Easy – it’s cell based, so it stays
on the plate• Biologically Relevant –phosphorylation state of regulatory
sites is quickly characterized
A variety of kits to choose from
FACE Kits are available for over 20 different targets covering the major signaling
pathways (see web address below). The
Suspension Cell FACE module works
with all FACE Kits; it improves results
when working with suspension cells by
providing 96-well ﬁlter plates that makeit easier to perform washing & liquidhandling steps. And, with FACE Maker
Kits, you can use your own primary andsecondary antibodies to detect any
target or modiﬁcation state of interest.
• With FACE™ NFκB p65 Proﬁler,
distinguish between p65 that is
phosphorylated at Ser468 vs. Ser536• Detect the anti-apoptotic
phosphorylation of Bad at Ser112• Characterize the activation of the
MAP kinases ERK1 and ERK2
• Proﬁle the JAK-STAT pathway:
JAK1, STAT2, STAT4, STAT6• Study the receptor tyrosine kinases
EGFR and HER-2• Monitor c-Jun phosphorylation at
Ser63 or Ser73
Simplify your phospho-assays today!
For complete information on FACE,
please visit www.activemotif.com/face.
To learn how to get FACE Kits at 30% off,
visit www.activemotif.com/promo.

Stimulatecells

Flow chart of the FACE process.

relative phosphorylation state, and areadout of the activation state of thatsignaling pathway (Figure 1).

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9

SUMOylation & Luciferase Reporter Assays

SUMOlink™ Enables Efﬁcient Analysis of the Effects of SUMOylation
SUMOlink™ Kits provide a fast, simple method for generating SUMOylated proteins in vitro. SUMO (small ubiquitinlike modiﬁer) shares degrees of sequence identity with ubiquitin, a post-translational modiﬁcation that serves avariety of functions, such as protein sorting, transcriptional activation and DNA repair. The ability to investigate theeffects of SUMOylation on enzyme function may help in understanding the role of SUMOylation in the regulation ofcellular processes and the identiﬁcation of novel proteins as targets for SUMO.
Why use SUMOlink?
• Simple, effective method for SUMO
conjugation and detection of
SUMOylated proteins• Positive control p53 protein andantibody provided to ensure success• Wild-type and mutant SUMO
proteins are included• Versatile – study SUMOylation ofcell or tissue extracts, or recombinant proteins

The SUMOlink method
With SUMOlink, simply add the assay
components to a microcentrifuge tube
with your protein of interest. Aftera 3-hour incubation, the reaction is
stopped and results can be analyzed byWestern blot. With the kit’s SUMO-1 or
SUMO-2/3 antibodies, you can easily see
the extent to which your target protein
has been SUMOylated. The included p53
protein and antibody serve as a positive
control, while mutant SUMO proteins
are included as a negative control.
To learn more, please call or visit us atwww.activemotif.com/sumolink.

What’s in the box?
Each kit contains E1 activating and E2
conjugating enzymes along with wildtype and mutant SUMO-1 (SUMO-1
Kit) or SUMO-2 and SUMO-3 proteins(SUMO-2/3 Kit). Antibodies for SUMO1 or SUMO-2/3 modiﬁcations, as wellas control p53 protein and antibody.
Enough reagents are provided toperform 20 in vitro SUMOylations withthe wild-type protein and 20 in vitro
SUMOylations with the mutant protein.

Product

FormatCatalog No.

SUMOlink™ SUMO-1 Kit

20 rxns

40120

SUMOlink™ SUMO-2/3 Kit

20 rxns

40220

Sensitive Reporter Assay for Better Screening Results
Active Motif’s patented RapidReporter® gene assays provide a faster, more pronounced response to stimulation andrepression than other systems. RapidReporter vectors include double destabilization elements for both the luciferase
protein and its mRNA, so the assay yields more accurate kinetic and drug concentration-dependent responses andenables researchers to detect smaller changes in activity than is possible with non-destabilized reporter gene assays.

10

North America 877 222 9543

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Why use RapidReporter?
• Stronger fold induction reduces
false positives• Faster response enables detectionof transient effects, and drugs that
decompose rapidly (Figure 1)• Your choice of stringency –pRR-High for best response tochanges in transcription andpRR-Low for a stronger basal signal• “Empty” vectors available for cloning your own elements in, or use
pre-made vectors containing widely
studied promoter sites

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Figure 1: RapidReporter “unmasks” hidden effects.
HeLa cells were transiently transfected with pRR-High
NFκB or pGL3 vector containing NFκB, then plated in
96-well plates. Twenty-four hours post-transfection,
the cells were stimulated with 10 ng/ml TNF-α andmeasured for Gaussia Luciferase (pRR) and firefly
luciferase (pGL3) at the indicated time points. Because
RapidReporter has double destabilizing elements that
reduce background, relatively small and transient events,
like the natural oscillation of NFκB from the cytosol to
the nucleus during its activation, can be observed.

RapidReporter is available in a variety of formats to suit your research needs. You canpurchase either luciferase assay reagents, vectors or the vector and assay reagents
together. For complete information about RapidReporter and a list of available products,
please visit www.activemotif.com/rapidreporter.

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www.activemotif.com

October 2010 • volume 11 • number 3Competent E. coli & Protein Delivery

Efﬁcient, Extremely Affordable Competent E. coli in a Convenient Format
RapidTrans™ are high-efﬁciency competent E. coli supplied in a convenient
96-tube tray. Ideal for cloning, plasmid preparation and library construction,
RapidTrans cells are affordably priced and packaged in a format that provides
maximum ﬂexibility while eliminating waste. Each tube contains 50 µl of cells
for one transformation reaction. This enables the use of as few or as many
reactions as needed, without thawing the other cells. This eliminates wasted
cells and the reduced efﬁciencies caused by repeated freeze/thaw cycles.
Use only what you need
Unlike other 96-well formats, which
force you to thaw all 96 wells, RapidTransis a tray of 12 x 8 tubes that can be usedsingly or with multi-channel pipettors.
So, you can thaw only what you’ll use.
Don’t pay for unused capacity
Compared to other suppliers, RapidTransare very affordable, primarily because
the transformation efﬁciency of TAM1
E. coli is slightly less, at > 1 x 108 cfu/µg

supercoiled pUC19 DNA. However, this is
more than adequate for most uses. So,
don’t pay more for super competency
cells that impact your lab’s budget, but
not your results. You wouldn’t buy a
Ferrari to go to the supermarket, wouldyou? Spend wisely; try RapidTrans today!

TAM1 genotype and contents

mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15∆lacX74 recA1 araD139 ∆(ara-leu)7697 galU
galK rpsL endA1 nupG

For your convenience, cells include SOC
media, pUC19 DNA and sterile reservoirs
for use with multi-channel pipettors.

Product

FormatCatalog No.

RapidTrans™ TAM1 Competent E. coli

1 x 96 rxns
5 x 96 rxns

11096
11596

Chariot™ Delivers Functional Protein Directly into Living Cells
The Chariot™ delivery reagent efﬁciently transports biologically active proteins, peptides and antibodies directly
into cultured mammalian cells. Delivery is complete in less than two hours and provides efﬁciencies of 65-95%. Afterdelivery, living cells can be assayed immediately to determine the effects of the introduced material. These features
make Chariot an ideal tool for a variety of functional studies.
Targeted delivery of functional protein
Chariot is a peptide that forms a noncovalent complex when incubated with
puriﬁed protein, peptide or antibody
for 30 minutes at room temperature.
Adding the complex to cells results in
its rapid internalization. Once inside thecell, the complex dissociates and Chariot
is transported to the nucleus, while thedelivered protein is biologically active
and free to proceed to its cellular target.

macromolecule. However, this canchange the folding characteristics ofyour protein and, ultimately, its function.
Because Chariot forms a non-covalentbond with the protein, it does not affectthe delivered protein’s folding or function.

Non-covalent delivery
Many protein delivery systems requirethat you fuse a carrier protein to your

Product

FormatCatalog No.

Chariot™

25 rxns
100 rxns

30025
30100

www.activemotif.com

What can Chariot do for you?
Direct delivery of active protein makes
it easy to perform many studies that are
not possible using DNA transfection andJapan +81 (0)3 5225 3638

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expression. Chariot results have been extensively published; successful deliveryof proteins, peptides and antibodies has
been shown in a wide range of cell lines,
including hard-to-transfect neuronal,
primary and plant cells. The method has
proven to be effective on both adherent and suspension cells, as well as invivo. For a more complete information,
including a list of papers citing Chariot,
go to www.activemotif.com/chariot.

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11

Trying to solve the puzzle ofHistone Modiﬁcations?
Why not send for our
New Poster? (see page 2)