THE NEWSLETTER OF ACTIVE MOTIF — February 2010 • volume 11 • number 1

Do
Not
Miss
This!
Active Motif’s new DNA Methyltransferase Assaychanges everything....(see page 2)

IN
THIS
ISSUE

2 NEW: Non-radioactive Assay to Screen
for DNA Methyltransferase Activity

7 Our Antibody Development Process Helps
Ensure Our Products Perform as Speciﬁed

3 NEW: A Better Way to Screen forProtein:DNA Binding

8 Chromeo™ Py-Dyes Eliminate Backgroundin Fluorescent Protein Labeling

3 Improved Yields of Nuclear, Cytoplasmic
or Whole-cell Extracts

9 Fluorescent Secondary Antibodies forEnhanced Detection

4 NEW: Simple Histone ELISAs to
Detect Mitotic Markers

10 NEW: Amplify Genomic DNA Samples
Without Representational Bias

4 HiLite™ Fluorescence Polarization
Histone Binding Assays

10 Recombinant Transcription Factors for the
Study of Regulatory Mechanisms

5 Easily Reconstitute Chromatin with the
Chromatin Assembly Kit

11 ELISA Method for More Accurate
Quantiﬁcation of Activated Ras GTPase

5 NEW: Recombinant Core Histone Proteins
with Site-speciﬁc Methylation

11 Method to Measure the Activity of
DNA Repair Proteins

6 Ensure Your Research Success with
Antibodies Proven to Work in ChIP

DNA Methyltransferase Activity Assay

NEW: Non-radioactive Assay to Screen for DNA Methyltransferase Activity
Looking for a fast, non-radioactive alternative to screen puriﬁed enzymes or nuclear extracts for DNA
methyltransferase activity or inhibition? Active Motif’s DNMT Activity / Inhibition Assay utilizes the high afﬁnity of
methyl CpG binding domain (MBD) proteins towards methylated DNA to provide a highly sensitive assay capable ofdetecting DNA methyltransferase activity and/or inhibition from puriﬁed DNMT1, DNMT3a & DNMT3b enzymes or
nuclear extracts. This simple colorimetric assay can be completed in less than 3 hours.

Active Motif also offers DNA methylation antibodies and enrichment kits. For
more information, please call or visit us
at www.activemotif.com/dnamt.

2

North America 877 222 9543

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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.

What comes in the kit?
The DNMT Activity / Inhibition Assay
contains a universal CpG-enriched DNA
substrate coated to a 96-stripwell plate.
Puriﬁed DNMTs or DNMT activities from
nuclear extracts will catalyze the transferof methyl groups from the provided
AdoMet reagent to the coated DNA substrate. A His-tagged recombinant MBD2b
protein is used to bind the newly methylated DNA substrate with high afﬁnity.
Addition of a polyHistidine antibody
conjugated to horseradish peroxidase(HRP) and developing solutions provide
a sensitive colorimetric readout that is
easily quantiﬁed by spectrophotometry.
A small sample of puriﬁed DNMT1
enzyme is included as a positive control.

Screen for inhibitors
Screen for DNA methyltransferase
inhibitors with the DNMT Activity /Inhibition Assay. DNA methyltransferase
inhibitors can provide key insight into
understanding gene regulation, especially as it relates to hypermethylationof tumor suppressor genes. The stripwellplate offers the convenience to perform
either low- or high-throughput screening. To demonstrate the effectivenessof screening for DNMT inhibitors, MCF-7
cells that were either untreated ortreated with the DNA methyltransferase
inhibitor procaine for 96 hours were
tested in the DNMT Activity / Inhibition
Assay (Figure 2).
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DNMT Assay advantages• Non-radioactive – colorimetricassay is easily quantiﬁed by spectrophotometry on a microplate reader
at 450 nm• Sensitive – unique MBD protein
approach enhances the sensitivityof detection from either puriﬁedproteins or nuclear extracts• Fast – can be completed in less than
3 hours• Less effort required – the kit iscompatible with multi-channel
pipettors to streamline wash steps• Flexible – stripwell plate allows
screening in low or high throughput

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Get improved sensitivity
Active Motif’s new DNMT Activity /Inhibition Assay is a fast, user-friendly
assay to speciﬁcally detect DNA
methyltransferase activity from recombinant DNMT enzymes or nuclear extract
samples without the need for radioisotopes or expensive equipment. This
assay is unique in that it utilizes a methyl
CpG binding domain protein (MBD) todetect methyltransferase activity. MBD
proteins are capable of binding methylated DNA with a higher afﬁnity thanantibody approaches, which increases
the sensitivity of the assay. With thismethod, as little as 0.5 ng of puriﬁed
enzyme (Figure 1) or 0.5 µg of nuclear
extract can be detected.

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C9<#-Y[bbi!%#/,^ekhFheYW_d[jh[Wjc[djFigure 2: DNMT inhibition with procaine treatment.
The DNMT Activity / Inhibition Assay was used toscreen for DNMT inhibition in MCF-7 cells that were
either untreated or treated with procaine for 96 hours.
Nuclear extracts were prepared using Active Motif’s
Nuclear Extract Kit (Catalog No. 40010); 5 µg of eachtreatment condition was tested in the assay with a
1.5 hour incubation time and a 3 minute developing time.
The 5 mM procaine treatment showed a 43% inhibition
of DNMT activity as compared to mock treated sample.

Product

FormatCatalog No.

DNMT Activity / Inhibition Assay

1 x 96 rxns

55006

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

February 2010 • volume 11 • number 1

EMSA & Nuclear Extraction Assays

NEW: A Better Way to Screen for Protein:DNA Binding
The GelShift™ Chemiluminescent EMSA Kit simpliﬁes the process of identifying protein:DNA interactions by providing
an easy-to-use assay with proven reagents. In this electrophoretic mobility shift assay (EMSA), cell extracts or puriﬁed
factors are incubated with biotin end-labeled probe containing the consensus binding site of interest; these binding
reactions are then resolved 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
GelShiftP
Digoxigenin
Chemi EMSA
LabeledLabeled
Exposure Time
3 minutes
16 hours
60 minutes
DNA band. The non-radioactive format does not sacriﬁce sensitivity whencompared to 32P or digoxigenin-labeled methods (Figure 1).
32• Non-radioactive assay• Includes control DNA and extract• Fast procedure can be completed in 5 hours• Additional reagents supplied to help optimize conditions for yoursample system
Product

FormatCatalog No.

Gelshift™ Chemiluminescent EMSA

100 rxns

37341

HeLa Extract
Competitor

- + +
- - +

- + +
- - +

- + +
- - +

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

Improved Yields of Nuclear, Cytoplasmic or Whole-cell Extracts
Active Motif’s Nuclear Extract Kit isolates high yields of active nuclear, cytoplasmic or whole-cell extract frommammalian cell or tissue samples. The simple protocol has been optimized to ensure that proteins remain
segregated in their particular fraction, which eliminates cross-contamination that can cause misleading results.
Why use the Nuclear Extract Kit?
Extracts prepared using the Nuclear
Extract Kit can be used in any procedurethat requires nuclear, whole-cell or cytoplasmic extract, including Active Motif’s
TransAM Transcription Factor DNAbinding assays (Figure 1). The high yields
and speciﬁcity of protein fractions help
you save time and allow you to achieve
results faster and more accurately.

Nuclear Extract Kit advantages• Complete kit contains all required
reagents, including protease andphosphatase inhibitors• No need for optimization• Quality controlled reagents ensure
reproducibility• Ability to prepare extracts fromboth cultured cells and tissue
samples

Product

FormatCatalog No.

Nuclear Extract Kit

100 rxns
400 rxns

40010
40410

Figure 1: Speciﬁc nuclear and cytoplasmic extraction.
Nuclear and cytoplasmic extracts were prepared using
the Nuclear Extract Kit from HeLa samples unstimulated
or stimulated with TNF-α for 30 minutes and assayedusing the TransAM™ NFκB p50 Kit. Because activated
NFκB translocates to the nucleus, only nuclear extractfrom stimulated cells should contain activated NFκB.

Special Offer:
Through the end of March 2010, purchase any of Active Motif’s TransAM™ Transcription Factor DNA-binding Assays and receive a
FREE Nuclear Extract Kit. Simply cite promo code SP2-2010 when you place your order. Visit www.activemotif.com for more details.

www.activemotif.com

Japan +81 (0)3 5225 3638

Europe +32 (0)2 653 0001

North America 877 222 9543

3

Histone Modification ELISAs & Histone Tail Binding Assay

NEW: Simple Histone ELISAs to Detect Mitotic Markers
Active Motif has expanded its line of Histone Modiﬁcation ELISAs to include assays that detect phosphorylation ofserine 10 or serine 28 on histone H3, which serve as markers for cells undergoing mitosis. We have applied ourexpertise in making histone modiﬁcation antibodies to produce optimal antibody pairs for the detection of
speciﬁc histone modiﬁcations in an easy-to-use sandwich ELISA format.
Other Histone Modiﬁcation ELISAs
Active Motif also offers quantitative
Histone ELISA Kits to detect methylation
events on histone H3. Methylation oflysine residues in nucleosomal histones isthought to mediate interactions with theprotein complexes involved in regulatingtranscription, replication and DNA repair.
These assays include our unique recombinant histone H3 proteins (page 5) withsite- and degree-speciﬁc methylation foruse as a reference standard curve.

For more information about our Histone
Modiﬁcation ELISAs, please call or visit
www.activemotif.com/hiselisa.

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How do the Histone ELISAs work?
The Histone Modiﬁcation ELISA Kits
include everything necessary to screen
puriﬁed core histones or acid extracts
for changes in phosphorylation levels.
A 96-stripwell plate is coated with ahistone H3 antibody to capture histone
H3 from the sample, and an antibody
that detects phosphorylation ateither serine 10 or serine 28 is added forspeciﬁc detection. An HRP-conjugatedsecondary antibody and developing
solutions provide a sensitive colorimetric readout that is easily quantiﬁed byspectrophotometry (Figure 1). For added
convenience, positive and negative
control acid extracts are included.

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Figure 1: Histone H3 phospho Ser28 sensitivity.
Untreated and paclitaxel treated HeLa cells were acidextracted (Catalog Nos. 36200 & 36201) and tested
for changes in phosphorylation levels of serine 28 on
histone H3 from as little as 78 ng of acid extract.

Product

FormatCatalog No.

Histone H3 phospho Ser10 ELISA

1 x 96 rxns

53111

Histone H3 phospho Ser28 ELISA

1 x 96 rxns

53100

Histone H3 trimethyl Lys27 ELISA

1 x 96 rxns

53106

Histone H3 dimethyl Lys9 ELISA

1 x 96 rxns

53108

Histone H3 trimethyl Lys9 ELISA

1 x 96 rxns

53109

Total Histone H3 ELISA

1 x 96 rxns

53110

HiLite™ Fluorescence Polarization Histone Binding Assays
Active Motif’s new HiLite™ Binding Assay uses ﬂuorescence polarization to provide researchers with an innovative,
high-throughput tool for monitoring interactions between histone tails and chromatin-modifying proteins. The
unique format of this assay enables homogenous quantitation of biologically important transcriptional regulation
events, and can be used to determine the speciﬁcity of a particular protein for methylated histone tail peptides.
HiLite Binding Assay advantages• Homogeneous – no washing steps
are required• Fast – reactions reach equilibrium injust seconds to minutes• Reproducible – kit reagents arestable and prepared at one time,
resulting in high reproducibility• Robust – resistant to changes in pH,
temperature, salt concentration, etc.

4

North America 877 222 9543

With Active Motif’s HiLite Histone H3
Methyl-Lys9 / Lys27 Binding Assay, you
can use ﬂuorescence polarization todetermine if your speciﬁc protein ofinterest binds histone H3 that is methylated at either lysine 9 or 27. You canalso measure the afﬁnity of the binding

interactions between your protein ofinterest and speciﬁc histone methylation
states, which in turn enables fast andefﬁcient inhibitor screening studies.
For additional details, please visit us atwww.activemotif.com/hilite.

Product

FormatCatalog No.

HiLite™ Histone H3 Methyl-Lys9 / Lys27 Binding Assay

1 kit

57001

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

February 2010 • volume 11 • number 1

Chromatin Assembly & Recombinant Histones

Easily Reconstitute Chromatin with the Chromatin Assembly Kit
Active Motif’s Chromatin Assembly Kit makes it easy for you to assemble chromatin on your linear or supercoiled
DNA of interest to investigate gene regulation in a context that closely resembles in vivo chromatin. The kit contains
proven reagents that provide an easy and complete solution for chromatin generation.
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 thekit components with your DNA• Produces an excellent substrate forvarious gene regulation experiments

promoter in chromatin. Thus, using DNA
not assembled into chromatin for testing
can generate misleading results.

Why assemble chromatin?
Research has indicated that the transcriptional regulation of a promoter on
naked DNA differs from that of aWith the Chromatin Assembly Kit, theATP-dependent method utilizes puriﬁed
recombinant human chromatin assemblycomplex ACF and histone chaperone
NAP-1 with puriﬁed HeLa core histones
for in vitro assembly of extended,
regularly ordered, periodic arrays of
nucleosomes (Figure 1). This enables you
to mimic the chromatin environment,
so you can study regulation underconditions that are closer to real life.

Product

FormatCatalog No.

Chromatin Assembly Kit

10 rxns

53500

HeLa Core Histones

36 µg

53501

M

1

2

3

4

Figure 1: Enzymatic digestion of assembled chromatin.
Limited micrococcal nuclease digestion was carried out
on chromatin assembled using 1 µg samples of circular
DNA (Lanes 1 & 2) or linear DNA (Lanes 3 & 4), resulting
in regularly spaced nucleosomes.

NEW: Recombinant Core Histone Proteins with Site-speciﬁc Methylation
Post-translational modiﬁcations on histone tails have been shown to correlate very closely with the nuclear processesof transcription, chromosome packaging and DNA damage repair. Active Motif offers recombinant histones withspeciﬁc methylated lysine residues to better investigate how methylation patterns impact regulatory processes.
How are they made?
Recombinant methylated histones are
created using a patented approach inwhich an analog of methyl lysine isincorporated into the histone via achemical alkylation reaction. This
enables the site and degree of methylation to be carefully controlled. Because
the engineered methylation state closely
mimics natural methylation, theserecombinant proteins are ideal for use
in functional assays.

Product

FormatRecombinant Histone H2A NEW

50 µg

Recombinant Histone H2B COMING SOON

50 µg

Recombinant Histone H3 (C110A)

50 µg

Recombinant Histone H3 Lys4 (mono-, di-, or trimethyl)

50 µg

Recombinant Histone H3 Lys9 (mono-, di-, or trimethyl)

50 µg

Recombinant Histone H3 Lys27 (mono-, di-, or trimethyl)

50 µg

Recombinant Histone H3 Lys36 (mono-, di-, or trimethyl)

50 µg

Recombinant Histone H3 Lys79 (mono-, di-, or trimethyl)

50 µg

Recombinant Histone H4

50 µg

Recombinant Histone H4 Lys20 (mono-, di-, trimethyl)

50 µg

For more complete information, please
visit www.activemotif.com/mehisprots.

www.activemotif.com

Japan +81 (0)3 5225 3638

Europe +32 (0)2 653 0001

North America 877 222 9543

5

ChIP-validated Antibodies

Ensure Your Research Success with Antibodies Proven to Work in ChIP
ChIP can be technically challenging and difﬁcult to validate without well-proven reagents. By providing proven
ChIP-validated antibodies, reagents and controls, Active Motif can help you achieve the best results possible.
Chromatin immunoprecipitation (ChIP)
is a powerful and important techniquethat allows researchers to identify thein vivo localization sites of a chromatinprotein or transcription factor, or toﬁnd regions of the genome associatedwith a particular histone modiﬁcationor histone variant. In combination withmicroarray methods or next generation
DNA sequencing, ChIP allows some very
important questions to be asked on a
genome-wide scale.
While the number of commerciallyavailable antibodies is growing on a daily
basis, few companies can validate theirantibodies for use in ChIP. This meansthat scientists often spend considerable
amounts of time and money validating antibodies. Active Motif specializes in manufacturing high-quality
ChIP-validated antibodies to histones,
histone modiﬁcations, chromatin proteins and transcription factors. When
you buy one of our ChIP-validated
antibodies, you can be conﬁdent that it
will work for you because our in-housescientists have validated it (Figures 1-2).
Competitive Testing
Our antibodies stack up against thecompetition, and we can prove it. To see
how some of our antibodies to important histone modiﬁcations compared tothose of our competitors, we put themthrough side-by-side comparison testing.
Identical ChIP experiments were performed using the amount of antibody
recommended by each manufacturer.
Real-time quantitative PCR was carriedout using primers speciﬁc for the geneindicated, and the results were plotted
as “Fold Enrichment” relative to an IgG
negative control (Figures 3-5).

6

North America 877 222 9543

The table below list some of Active
Motif’s ChIP-validated antibodies
directed against histone modiﬁcations.
For a complete list of all of antibodies
validated for use in ChIP, please visit us
at www.activemotif.com/chipabs.

Figure 3: Competitive testing of ChIP-validated
antibodies against Histone H3 trimethyl Lys27.

Figure 1: ChIP analysis of Histone H3 dimethyl Lys9 pAb.
Chromatin IP performed using the ChIP-IT™ Express Kit(Catalog No. 53008) and HeLa Chromatin (1.5 x 106 cell
equivalents per ChIP) using 10 µl of Histone H3 dimethyl
Lys9 pAb (Catalog No. 39375) or the equivalent amountof rabbit IgG as a negative control. Real time, quantitative PCR (RT-qPCR) was performed on DNA purified
from each of the ChIP reactions using a primer pair
specific for either the PABPC1 gene or the MyoD gene.

Figure 2: Histone H3 trimethyl Lys27 pAb tested by ChIP.
ChIP performed using the ChIP-IT™ Express Kit and 50 µlof Ready-to-ChIP HeLa Chromatin (Catalog No. 53015)
per ChIP. Following the ChIP reaction, DNA was purified
from the immunoprecipitated chromatin and a region
approximately 1000 base pairs 5´ of the transcriptional
start site of the HoxD13 gene was amplified by PCR.
Lane 1: ChIP using negative control rabbit IgG. Lane 2:
PCR input control. Lane 3: ChIP using 10 µl of Histone H3
trimethyl Lys27 pAb (Catalog No. 39155).

Product

Figure 4: Competitive testing of ChIP-validated
antibodies against Histone H3 trimethyl Lys9.

Figure 5: Competitive testing of ChIP-validated
antibodies against Histone H4 acetyl Lys16.

FormatCatalog No.

Histone H3 monomethyl Lys4 mAb (Clone MABI 0302)

100 µg39635

Histone H3 dimethyl Lys4 mAb (Clone MABI 0303)

100 µg39679

Histone H3 monomethyl Lys9 mAb (Clone MABI 0306)

100 µg39681

Histone H3 dimethyl Lys9 mAb (Clone MABI 0307)

100 µg39683

Histone H3 acetyl Lys27 mAb (Clone MABI 0309)

100 µg39685

Histone H3 trimethyl Lys27 mAb

100 µg39535

Histone H4 trimethyl Lys20 mAb (Clone 6F8-D9)

100 µg39671

Europe +32 (0)2 653 0001

Japan +81 (0)3 5225 3638

www.activemotif.com

February 2010 • volume 11 • number 1

Antibody Development & Validation

Our Antibody Development Process Helps Ensurethat Our Products Perform as Speciﬁed
At Active Motif, antibody development and testing is a very important andtightly controlled process, with many steps along the way. From the designof the immunogen to the speciﬁcity screening and application validation,
our years of expertise in antibody development ensures that only thehighest quality antibodies are offered for use in your research.
Immunogen selection
Immunogens are selected to decrease the likelihood of cross-reactivity with related
proteins, and to maximize detection of the protein in its native context. Immunogens
for modiﬁcation-speciﬁc antibodies are
selected to ensure that the antibody
recognizes only the modiﬁed version of theprotein, and does not cross-react with thesame modiﬁcation on different proteins.
Speciﬁcity screening
The ﬁrst test performed on every antibody
is dot blot analysis, which ensures its speciﬁcity for the desired protein or modiﬁcation(Figure 1). Antibodies that do not exhibit agreater than 25-fold selectivity for thedesired modiﬁcation are failed.

Figure 1: Dot blot analysis conﬁrms speciﬁcity.
10, 50 and 250 picomole aliquots of synthetic peptides
corresponding to histone H3 that is unmodified, mono-,
di- or trimethylated at Lys4 (Lanes 1-4), Lys9 (Lanes 5-8)
or Lys27 (Lanes 9-12) were spotted and probed with
Histone H3 trimethyl Lys27 pAb (Catalog No. 39155),
confirming the antibody’s specificity for histone H3
trimethyl Lys27 (Lane 12). No cross-reactivity is observed
for the unmodified Lys27 peptide (Lane 9) and only ahint of signal is observed at the 250 picomole spots formono- (Lane 10) and dimethyl Lys27 (Lane 11).

Western blot
Western blotting is performed to verify thatthe antibody recognizes a protein of thecorrect molecular weight and does notcross-react with other proteins. Commonlyused chemical treatments that stimulate a
speciﬁc modiﬁcation (e.g. HDAC inhibitors
and acetylation) are used to better detect the modiﬁcation (Figure 2).

Application validation
Each antibody is validated for use in important applications such as IF (Figure 3) and ChIP
(Figures 1 and 2, page 6), so you can have conﬁdence when using them in your experiments.
Additional speciﬁcity testing
Antibodies to histone modiﬁcations that exist in budding yeast (S. cerevisiae) can befurther screened for speciﬁcity by testing strains of yeast that contain a point mutation
at the position of the modiﬁed amino acid (Figure 4).

For a complete listing of all of our high-quality antibodies,
please visit us at www.activemotif.com/abs.
www.activemotif.com

Japan +81 (0)3 5225 3638

Europe +32 (0)2 653 0001

Figure 2: Western blot conﬁrming size and modiﬁcation.
Histone H2B acetyl Lys120 pAb (Catalog No. 39119) wastested by Western blot. No reactivity is observed onrecombinant human histone H2B (Lane 1, 200 ng protein),as it is not acetylated. Lanes 2 and 3 contain 5 µg eachof HeLa cell acid extract. Cells that were treated with ahistone deacetylase inhibitor (sodium butyrate) prior to
extraction have an increased signal (Lane 3), which is to
be expected for an antibody to a site of acetylation.

Figure 3: Immunoﬂuorescence testing.
HeLa cells stained with alpha Tubulin mAb in red(Catalog No. 39527, panels A and D), Histone H4monomethyl Lys20 pAb in green (Catalog No. 39175,
panels B and D) and DAPI in blue (panels C and D).
Note the strong nuclear staining and absence of cytoplasmic staining in panel B, which is to be expected ofan antibody directed against a histone modification.

Figure 4: Mutational analysis of yeast modiﬁcations.
Histone H4 acetyl Lys12 pAb (Catalog No. 39165) wastested by Western blot using extracts from wild-type
yeast or yeast containing a mutated histone H4 gene.
Lane 1: Yeast with wild-type histone H4.
Lane 2: H4 with the amino-terminal tail deleted.
Lane 3: H4 with an arginine instead of lysine 5.
Lane 4: H4 with an arginine instead of lysine 8.
Lane 5: H4 with an arginine instead of lysine 12.
Lane 6: H4 with an arginine instead of lysine 16.
Lane 7: H4 with arginines instead of lysines 5, 8 & 12.
Note the absence of reactivity in yeast extracts in which
lysine 12 is either missing or mutated (Lanes 2, 5 & 7).

North America 877 222 9543

7

Fluorescent Labels & Stains

Chromeo™ Py-Dyes Eliminate Background in Fluorescent Protein Labeling
Py-Dyes represent a new class of ﬂuorescent pyrylium molecules that undergo a shortwave spectral shift and alarge increase in quantum yield when they react with amine groups on proteins. As a result, their ﬂuorescence is only
detectable when the dye is conjugated to protein, which eliminates background. In addition, Py-Dyes have a numberof other properties that make them ideal for labeling proteins. Five different Py-Dyes are available, and several
staining kits that utilize Py-Dyes are also offered.
Variety of Py-Dyes available
Table 1 shows the Py-Dyes available andtheir physical properties. In every case,the conjugated dye is the sole emitter at
the speciﬁed wavelength, ensuring thelack of background described above. For
complete information on each Py-Dye,go to www.activemotif.com/pydye.
absorb (503 nm)

emit (600 nm)

absorb (612 nm)

1.0

Fluorescence excitation

Advantages of Py-Dye protein labeling• Py-Dyes are supplied ready to use,with no need for activation• Conjugation is a simple, one-step,room-temperature incubation• Labeled protein can be used
immediately after conjugation• Because unreacted Py-Dye ishydrolyzed during conjugation,
there is no need for puriﬁcation,and background is eliminatedUnbound
Conjugated

0.8
0.6
0.4
0.2

emit (665 nm)

0
400 450 500 550 600 650 700 750 800

Wavelength (nm)Figure 1: Spectral shift following conjugation.
The emission of Chromeo P503 in its unbound, free stateis blue. Following conjugation to protein, the color ofthe dye changes to red.

Better chemistry yields better results
A useful property of Py-Dyes is thelarge spectral shift they undergo upon
conjugation, which greatly changes the
wavelength of their emission (Figure 1).
As shown in Figure 2, unbound Py-Dye
P503 has negligible emission. However,when conjugated to protein, it absorbs
at a much shorter wavelength and its
quantum yield increases dramatically.
When excited in the 488 nm range,
only conjugated P503 ﬂuoresces, and its
peak ﬂuorescence is easily distinguishedfrom that of Chromeo 488 and other488-excitable dyes.

8

North America 877 222 9543

Figure 2: Absorption and emission spectra offree vs. bound Chromeo P503.
Normalized absorption and emission spectra of
Chromeo P503 when the dye is free (solid, purple lines)
and conjugated (dotted, copper lines). As free dye andbound dye absorb at different wavelengths (612 nm vs.
503 nm) and the quantum yield of bound dye is 50-foldgreater than that of free dye, background from any free
dye that is not hydrolyzed is effectively eliminated.

Convenient staining kits
Active Motif has also utilized Py-Dyes as
the basis for several useful staining kits:• The Py-Dye in the Chromeo™ Live
Cell Mitochondrial Staining Kit
diffuses passively through themembrane of living cells, where it
localizes at the mitochondria. This
enables mitochondrial staining
without ﬁxation or washes.
• The Chromeo™ Red Fluorescent
Fixed Cell Staining Kit stains thenuclear membrane, mitochondria,ﬁbers and nucleoli in ﬁxed cells; it
can also be used as a counterstain in
immunoﬂuorescence experimentswith green dyes such as FITC orother 488-excitable dyes.
• The Py-Dye in the ProStain™ Protein
Quantification Kit reacts broadly
with all proteins, so its ﬂuorescenceintensity is directly proportional toprotein concentration.
For complete details on these kits,please visit us at www.activemotif.com.

Py-DyeDye State

Absorption

EmissionεL/(mol-cm)

Quantum
Yield*

Chromeo™ P465

Free
Conjugated

655 nm
465 nm732 nm
630 nm25,00025,000< 1%~14%Chromeo™ P503

Free
Conjugated

612 nm503 nm

665 nm
600 nm

60,00024,000< 1%~50%Chromeo™ P540

Free
Conjugated587 nm503 nm—
627 nm80,00050,0000%~20%Chromeo™ P429

Free
Conjugated

457 nm429 nm—
536 nm65,00075,0000%~10%Chromeo™ P543

Free
Conjugated

570 nm543 nm—
590 nm110,000
57,0000%~15%

Table 1: Properties of Chromeo Py-Dyes*The Quantum Yield will depend on the Dye-to-Protein Ratio (DPR) of the conjugated protein.

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February 2010 • volume 11 • number 1

Fluorescent Secondaries

Fluorescent Secondary Antibodies for Enhanced Detection
Active Motif offers an extensive number of high-quality ﬂuorescent dyes, including the Chromeo™ line of ﬂuorescentdyes, giving you a variety of choices for ﬂuorescent detection. Our proprietary dyes exhibit equivalent or superior
ﬂuorescent performance compared to existing ﬂuorophores. They have been developed speciﬁcally to be compatible
with all light sources, and match the most commonly used wavelengths in ﬂuorescent microscopes. For the utmost
in convenience, the dyes are available as conjugates to goat anti-mouse and goat anti-rabbit secondary antibodies,for immediate use in ﬂuorescent experiments. In addition to our pre-conjugated secondary antibodies, we also offer
complete, optimized labeling kits to simplify conjugation of Chromeo Dyes to primary antibodies and other proteins.
Chromeo Secondary Fluorescent
Antibody Conjugates enable sensitiveand speciﬁc detection in immunoﬂuorescence, high-content screening, ELISA,FRET applications and ﬂow cytometry.
They are compatible with most excitation sources including diode lasers,
LEDs, tungsten lamps and xenon arc
lamps. The dyes have been conjugatedto high-quality secondary antibodies
by an optimized conjugation method,
including subsequent puriﬁcation frominterfering substances.

alpha Tubulin mAb
Histone H2AX pS139 pAb++
Chromeo™ 642 anti-rabbit Chromeo™ 488 anti-mouse

DAPI Nuclear Stain

Figure 1: Concurrent staining using Chromeo 488 and Chromeo 642 ﬂuorescent secondaries HeLa cells.
HeLa cells were left untreated (top row) or treated with 100 µM etoposide for 6 hours (bottom row) and methanol fixed.
The histone variant H2AX was then stained by Histone H2AX phospho Ser139 rabbit pAb (Cat. No. 39117) and Chromeo 642
goat anti-rabbit IgG (Cat. No. 15044), while tubulin was visualized using alpha Tubulin mouse mAb (Cat. No. 39527) and
Chromeo 488 goat anti-mouse IgG (Cat. No. 15031). The nuclei were stained using DAPI and the 3 separate images merged.

Why use Chromeo Secondaries?
• Brightness – high ﬂuorescentintensity improves sensitivity• Limited photobleaching – enables
multiple exposures and increased
exposure times• pH stability – great for biologicalassays• Flexible formats – in addition tosecondary antibody conjugates, thedyes are available as NHS-Esters,
Carboxylic Acids, and as Biotin &Streptavidin conjugates

Even better when used together
As shown in Figure 1, Chromeo 488
and Chromeo 642 secondary antibody
conjugates produce intense ﬂuorescencethat is resistant to fading, facilitating
multiple exposures and superior quality
images. Table 1 shows the properties ofthe various conjugates. As many of theiremission spectra are well separated, they
can be used together for multi-color

Secondary Antibody

Absorption

EmissionεL/(mol-cm)

Stokes Shift

Chromeo™ 488 IgG

488 nm

517 nm73,00029 nm&$,

Chromeo™ 494 IgG

494 nm

628 nm55,000124 nm&$*Chromeo™ 505 IgG

505 nm

526 nm70,000

21 nm

&$(

&
)+&

Chromeo™ 546 IgG

545 nm561 nm

98,80016 nm

642 nm

660 nm180,000

18 nm

Table 1: Properties of Chromeo Secondary Antibody Conjugates.

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staining experiments. The large Stokes
shift of Chromeo 494 (Figure 2) enables
it to be used even with Chromeo 488
and other 488-excitable dyes that have
similar excitation peaks.
Get all the information today
For complete details on our secondary
antibody conjugates, dyes and labelingkits, go to www.activemotif.com/ﬂuor.
'

Chromeo™ 642 IgG

www.activemotif.com

All 3 images merged

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9^hec[e*/*[c_ii_ed

&$.

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+&&

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,+&

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.&&

.+&

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Figure 2: The absorption and emission spectra of
Chromeo 494 have peaks separated by 124 nm.

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Whole Genome Amplification & Recombinant Transcription Factors

NEW: Amplify Genomic DNA Samples Without Representational Bias
Active Motif’s new GenoMatrix™ Whole Genome Ampliﬁcation Kit allows you to accurately duplicate DNA from yourexperiments for downstream analysis techniques that require more DNA than is yielded initially.
35
30
25

Ct Value

With the advent of techniques thatallow important questions to be asked
on a genome-wide scale, experiments
do not always yield sufﬁcient quantities of DNA. The GenoMatrix Kit can beused as a stand-alone product to amplify
unsheared DNA, or with pre-sheared
samples generated through use of Active
Motif’s ChIP-IT™ Express, MethylCollector™ or UnMethylCollector™ Kits. The kit
utilizes a new approach that virtuallyeliminates ampliﬁcation bias, so the ampliﬁed material has the same sequence
representation as the material that you
started with (Figure 1).

20
15
10
5
0

BRCA1

BRCA2

CREB1

DHFR

FosB

MAS1

NEDD4

NFκB

Figure 1: Representative and quantitative DNA
ampliﬁcation without sequence bias.
Quantitative real-time PCR was carried out on DNA
samples using primers indicated below each data set
and the Ct value for each sample plotted. The DNA wasderived from an original DNA sample amplified using the
GenoMatrix Whole Genome Amplification Kit (red bars),
amplified with a competitor’s whole genome amplification kit (copper bars) or not subjected to whole genome
amplification (purple bars).

Product

FormatCatalog No.

GenoMatrix™ Whole Genome Ampliﬁcation Kit

50 rxns

58001

GenoMatrix Kit advantages• Bias Free Amplificationof genomic DNA, even fromsmall amounts of sample• Preserve the Original Sequence
representation of your starting
material• Obtain Enough DNA
for genome-wide applications that
require large amounts of DNA
The GenoMatrix™ Kit provides up to a
500-fold ampliﬁcation starting from as
little as 10 ng of any type of genomic
DNA. GenoMatrix Kits provide all
reagents needed to quickly and easily
perform genome-representative ampliﬁcations. For more information, please
visit www.activemotif.com/wga.

Recombinant Transcription Factors for the Study of Regulatory Mechanisms
When studying transcriptional regulation, because it is useful to study the function of a speciﬁc transcription factor
in reconstitution assays, it is of great value to have available high-quality recombinant transcription factor proteins.
Recombinant transcription factors can be used in many biological applications, including as substrates in biochemical
assays, as many of them are also substrates for important regulatory enzymes. Active Motif offers a complete line ofrecombinant transcription factors for your researchneeds. Many of them, including NFκB p50, NFκB p65,
p53, c-Fos, c-Jun, c-Myc, CREB and Sp1 proteins arevalidated for use as standards in Active Motif’s
TransAM™ DNA-binding ELISAs.
Active Motif Transcription Factor Categories• General Transcription Factors• Nuclear Receptors• NFκB Family• Transcriptional Activators

Flow chart of the TransAM process.

A complete list of available recombinant transcription factors can be found at www.activemotif.com/tfprot.
10

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February 2010 • volume 11 • number 1

GTPase Activation & DNA Repair Protein ELISAsELISA Method for More Accurate Quantiﬁcation of Activated Ras GTPase

In contrast, the Ras GTPase Chemi ELISA
uses Raf-RBD protein and antibodies ina 96-well format to capture and quantify the activated Ras in your sample.
This faster, more sensitive alternative
enables you to use less of your precious
sample, yet still detect low-level events.
In addition, because ELISAs provide morequantitative results than Westerns, thedata generated are more meaningful.
The Ras GTPase Chemi ELISA method
Because activated Ras binds the
Ras-binding domain (RBD) of the Raf
effector protein, Raf-RBD is used as aprobe to isolate activated Ras. The kit

contains a Raf-RBD protein fused to GST
and a 96-well, glutathione-coated assay
plate. GST-Raf-RBD is ﬁrst incubatedon the plate for one hour to immobilize this capture protein. Additionof sample to the plate results in thebinding of activated Ras to the Raf-RBD.
Primary antibody speciﬁc for Ras andan HRP-conjugated secondary are thenadded, followed by developing reagent.
The plate is then read on a luminometer,
providing a sensitive, quantitative readout of activated Ras GTPase (Figure 1).

Ras GTPase ELISA advantages• More sensitive – assay uses only
25 µg of extract, or 20-fold less thanpull-down/Western methods• Better results – quantitative data
makes it easier to compare results• Less effort – no need to perform IP,
run gels or develop Western blots
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Active Motif’s Ras GTPase Chemi ELISA Kit is the ﬁrst ELISA-based kit
designed to detect and quantify activated Ras GTPase. The method offers
several advantages over other commercially available kits, which require youto perform immunoprecipitation of Ras, followed by Western blotting.

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-%),

()%,
),
:\cc\okiXZk«^&n\cc,'

Figure 1: Quantiﬁcation of activated Ras GTPase.
Increasing amounts of whole-cell extract from HeLa
cells either unstimulated or stimulated 2 minutes with
5 ng/ml EGF are assayed using the Ras GTPase ELISA Kit.

Product
Ras GTPase Chemi ELISA Kit

FormatCatalog No.

1 x 96-well plate

52097

Method to Measure the Activity of DNA Repair Proteins

DNA Repair Protein Kit advantages• ELISA format eliminates gels,blotting and radioactivity• Up to 10-fold greater sensitivity
than gelshift assays• Ability to assay both cultured cells
and tissue samples• Quantitative results in < 5 hours

The DNA Repair Protein Kit method
DNA Repair Protein Kits are better thanWestern for detection of DNA repair
proteins. Each kit contains a 96-wellplate with multiple copies of an immobilized oligonucleotide containing a DNA
lesion. When cell extract is added toeach well, the repair protein of interest

Product

FormatCatalog No.

GTBP DNA Repair Kit

1 x 96-well plate5 x 96-well plates

51096
51596

Ku70/86 DNA Repair Kit

1 x 96-well plate5 x 96-well plates

51196
51696

RPA DNA Repair Kit

1 x 96-well plate5 x 96-well plates

51296
51796

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binds speciﬁcally to the oligonucleotide on the plate. Addition of primary
antibody, HRP-conjugated secondaryand developing solution yields an easily
quantiﬁed colorimetric readout (Figure 1).

A

GTBP
A

1.25
1
OD450 nmDNA Repair Protein Kits are DNA-binding ELISAs that detect DNA repair
protein activity in both mammalian tissue and cell culture extracts. The sensitive, speciﬁc assay is 10-fold more sensitive than gelshift, without the needfor gels or radioactivity. Quantitative results are achieved in under 5 hours.

0.75
0.5
0.25

B

0

Raji

PANC-1

B

JurkatFigure 1: Measuring GTBP activity in several cell lines.
Jurkat, PANC-1 and Raji nuclear extracts were assessed
for GTBP activity using Western blot (A) and the GTBPDNA Repair Protein Kit (B). The DNA Repair Protein Kit isclearly more quantitative and sensitive.

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