THE NEWSLETTER OF ACTIVE MOTIF — April 2008 • volume 9 • number 1

2 NEW: Suspension Cell FACE™ – Quantify Phospho-proteins in Non-adherent Cells
3 NEW:  Magnetic Beads Improve Co-IP of Both Nuclear and Whole-cell Complexes
4 Rapid and Efficient Comparison of Methylation in Various DNA Samples
5 Upgrade Your Chromatin IP Experiments using ChIP-IT™ Kits and Reagents

IN
THIS
ISSUE

6 NEW:  High-quality Antibodies to Histones and Histone Modifications
7 NEW:  Sensitive HDAC Assay Kits in Colorimetric and Fluorescent Readouts
8 DNA Damage Assay – an Easy Assay for DNA Damage Signaling and Repair
9 NEW:  MAX Stain™ Immunofluorescence Products for Better IF Experiments
10 Most Published Transcription Factor Assay
11 Isolate Pure, Separate Fractions of Histone H2A/H2B and H3/H4 While
Preserving Post-translational Modifications
12 Investigate SUMOylation with Efficient SUMOlink™
12 MethylDetector™ – Fast, Reproducible DNA Methylation Analysis

Get our new
2008 Epigenetics catalog(see card inside).

Phospho-protein Detection

NEW: Suspension Cell FACE™ – Quantify Phospho-proteins in Non-adherent Cells
Fast Activated Cell-based ELISA (FACE™) Kits provide a simple, sensitive method for detecting phosphorylation directly in the cell, without making extracts or running gels. The new Suspension Cell FACE module greatly improves resultswith suspension cells by providing greater flexibility for the study of over 20 different kinase and receptor targets.

The FACE method
In FACE, cells are grown in a 96-well plateand stimulated to induce the pathway
of interest. After stimulation, the cells
are fixed to preserve protein activationand modification states. Each well isthen incubated with a primary antibody
specific to the protein of interest. An
antibody that recognizes all forms of theprotein is included as well as an antibody
that recognizes the phosphorylatedsite of the target protein. This enables
you to compare the effect of stimulation in your cells on both total andphosphorylated protein (Figure 1).
80
Normalized Signal (Relative Percent)

FACE – the “in-cell” Western
FACE Kits have become a popularmethod for studying phosphorylation
because they are fast, easy to use andthe results generated are more specific,
quantitative & reproducible than more
traditionally used methods like radioactive kinase assays and Western blotting.
Active Motif has developed FACE assays
for over 20 different kinase and receptor
targets (Table 1), and each is available inboth chemiluminescent and colorimetric formats. However, until now, themethod was not as effective when using
suspension cells.

70
60
50
40
30
20
10
0

Untreated

PMA treated

Untreated

Total ERK 1/2

PMA treated

Phospho-ERK 1/2

Figure 1: FACE ERK1/2 Chemi with Suspension Cell FACE.
Suspension Cell FACE was used with the FACE ERK1/2
Chemi Kit to assay total and phosphorylated ERK1/2
using 50,000 Jurkat cells per well. The cells were serumstarved and treated with 100 ng/ml PMA (Phorbol
12-myristate 13-acetate) for 15 minutes. A three-foldincrease in phospho-ERK1/2 was detected in the assay.

Capture & measure more cells
The Suspension Cell FACE module wasdesigned to work with any of our FACE
Kits. It provides 96-well filter plates thatmake it easier to perform washing &liquid handling steps through use of an
appropriate vacuum manifold. Thiseliminates the loss of suspension cells
that can occur when performing fixation
and wash steps in normal 96-well plates.
So, you’re able to measure a greaternumber of cells, which makes the assay
better able to measure small effects.
Suspension Cell FACE advantages• Adapt any of the optimized colorimetric or chemiluminescent FACE
Kits for use with suspension cells• No cell extracts or immunoblotting• Compare phosphorylated vs. total
target with the 2 included antibodies• Less than 2 hours of hands-on time
A variety of kits to choose from
FACE kits are available for over 20 different targets (Table 1). And, with the
FACE Maker Kits, you can use your own
primary and secondary antibodies todetect any target or modification stateof interest. For complete information on
the FACE product line, including the new
Suspension Cell FACE, please give us acall or visit www.activemotif.com/face.

Product

FormatCatalog No.

Suspension Cell FACE™

2 x 96 rxns

48305

Suspension Cell FACE™ Chemi

2 x 96 rxns

48405

Table 1: The FACE™ Product Line
FACE™ AKT (S473)

FACE™ ATF-2 (T71)

FACE™ Bad (S112)

FACE™ c-Jun (S63)

FACE™ c-Jun (S73)

FACE™ c-Src (Y418)

FACE™ EGFR (Y845)

FACE™ EGFR (Y992)

FACE™ EGFR (Y1173)

FACE™ ErbB-2 (Y877)

FACE™ ErbB-2 (Y1248)

FACE™ ERK1/2 (T202/Y204& T185/Y187)

FACE™ FAK (Y397)

FACE™ FKHR (T24)

FACE™ GSK3b (S9)

FACE™ HSP27 (S82)

FACE™ JAK1 (Y1022/Y1023) FACE™ JNK (S473)

FACE™ Maker

FACE™ MEK1/2 (S217/S221) FACE™ NFkB Profiler(S468 & S536)

FACE™ p38 (T180/Y182)

FACE™ P13 Kinase

FACE™ STAT4 (Y693)

FACE™ STAT6 (Y641)

2

FACE™ STAT2 (S473)

Toll Free — 1 877 222 9543

April 2008 • volume 9 • number 1

Magnetic Co-Immunoprecipitation

NEW: Magnetic Beads Improve Co-IP of Both Nuclear & Whole-cell Complexes
The Universal Magnetic Co-IP Kit includes protein G-coated magnetic beads
that improve co-immunoprecipitation (Co-IP) by simplifying the IP and wash
steps, while providing lower background. The kit also includes reagents to
prepare both nuclear and whole-cell extracts, so you can study intact protein
complexes whether they were originally bound to DNA or in the cytoplasm.
Protein/protein interactions are often
studied using Co-Immunoprecipitation(Co-IP), in which a single antibody is used
to precipitate an entire protein complex.
Additional antibodies are then used inWestern blot to identify/verify othercomplex members (Figure 1).

Figure 1: Nuclear Co-IP of SRC-1 and ERa.
The Universal Magnetic Co-IP Kit was used to makenuclear extract from MCF-7 cells induced 1 hour with
10 nM Estradiol. IP was performed on 300 µg samples
using 2 µg of SRC-1 pAb, ERa pAb and rabbit IgG (as anegative control). Western blot was then performed
using the ERa pAb on 10 µg Input Extract (Lane 1), SRC-1
IP (Lane 2), ERa IP (Lane 3) and the rabbit IgG IP (Lane 4).

Co-IP of cytoplasmic & nuclear proteins
While Co-IP is often used to study cytoplasmic protein complexes, traditional
methods are not optimal for studying
DNA-binding proteins because nuclear
complexes are very fragile. This oftencauses them to be disrupted during
extraction. For this reason, in addition
to containing components for wholecell extractions, the Universal Magnetic
Co-IP Kit provides nuclear extraction
reagents that have been optimized topreserve nuclear protein complexes. The
kit’s Enzymatic Shearing Cocktail uses
DNA digestion to gently release thecomplexes from the DNA, so they areintact and ready for Co-IP.

www.activemotif.com

Simpler procedure, lower background
The Universal Magnetic Co-IP Kit includes protein G-coated magnetic beads,
which simplify Co-IP by enabling IP andwash steps to be performed in seconds,
rather than having to use centrifugation.
Moreover, because the beads have very
low non-specific binding, backgroundis reduced even while using a low-salt
Co-IP/Wash Buffer that helps maintainweaker complexes.
Universal Magnetic Co-IP Kit advantages• Magnetic beads simplify procedureand reduce background• Optimized extraction method maintains nuclear protein complexes• Preserve protein modifications

Figure 2: Detection of acetylated Histone H3.
HeLa nuclear extracts were made using the Universal
Magnetic Co-IP Kit and a traditional high-salt extraction
protocol, each supplemented with the 1 µM trichostatin
A, a deacetylase inhibitor. Five and ten µg samples ofeach extract were used in Western blot with Histone H3
acetyl rabbit pAb (Cat. No. 39139). Protein was detected
only in samples made using the kit’s gentle nuclear
extraction procedure.

Figure 3: Preservation of acetylation and methylation.
Nuclear extracts were made from HeLa cells treatedwith 500 nM trichostatin A for 20 hours using either
the Universal Magnetic Co-IP Kit (with its deacetylase
inhibitor) or traditional high-salt extraction. Five and tenµg samples of these extracts were used in Western blot
with Histone H3 acetyl pAb (Cat. No. 39139) and Histone
H3 trimethyl Lys4 pAb (Cat. No. 39159). The acetylated
protein was detected only in the sample made using thekit. Methylation was slightly better maintained in thesample made using the kit.

Complete kit for better results
The Universal Magnetic Co-IP Kit has
both nuclear and whole-cell extraction
reagents, so you can perform IP on all
types of protein complexes. The kit also
includes protein G-coated magnetic
beads, a unique Co-IP/Wash Buffer
as well as phosphatase, protease anddeacetylase inhibitors that preserve theintegrity of the proteins. Finally, the kit
includes a strong bar magnet, so you cantake advantage of the improved wash
and IP steps enabled by the magnetic
beads. This makes the Universal Magnetic Co-IP Kit a simple, flexible andcomplete solution for getting more fromyour Co-IP. To find out more, please give
us a call or visit us on the web atwww.activemotif.com.

Product

FormatCatalog No.

Universal Magnetic Co-IP Kit

25 rxns

54002

3

DNA Methylation

Rapid and Efficient Comparison of Methylation in Various DNA Samples
Active Motif’s MethylCollector™ Kit provides users with a fast and efficient protocol for isolating and comparing
CpG-methylated DNA from cell or tissue samples. MethylCollector uses a recombinant Methyl-binding protein(MBD2b) to capture DNA, rather than traditional antibody-based immunoprecipitations, which improves sensitivity.
MethylCollector advantages• Fast and easy protocol completed
in less than 4 hours• Flexible – enables detection
from 5 ng to 1 µg of DNA• Suitable for use with DNA fragmented by sonication or enzymatic
digestion• Positive control DNA and PCRprimers ensure success
The MethylCollector method
In the MethylCollector Kit method,
His-tagged recombinant MBD2b protein
specifically binds to CpG-methylated
DNA fragments that have been preparedby enzymatic digestion or sonication.
These protein-DNA complexes arecaptured with nickel-coated magnetic
beads and subsequent wash steps are
performed with a stringent high-salt
buffer to remove DNA fragments thathave little or no methylation. Ready-touse methylated DNA is then eluted fromthe beads (Figure 1). MethylCollector ishighly efficient, enabling analysis of themethylation state of any specific locus
on genomic DNA isolated from less than800 cells (~5 ng DNA).
Applications of MethylCollector
The highly specific isolation of methylated DNA by MethylCollector enables
many powerful applications, including
the rapid screening of the methylation
status of multiple loci in tumor tissue
or cells. It can also be used to detectchanges in DNA methylation in othersituations, such as normal cellular differentiation and aging. To find out more,
please visit www.activemotif.com.

4

Fully Methylated Jurkat DNA Control
Fully Methylated Jurkat DNA is now
available separately for use as a control
in your DNA methylation applications.
Whether you’re performing bisulfite
sequencing, methylation-specific PCR(MSP), MeDIP or using our MethylCollector or MethylDetector™ Kits (page 12),Fully Methylated Jurkat DNA is a convenient positive control for investigating
CpG dinucleotide methylation (Figure 2).
Fully Methylated Jurkat DNA is suppliedwith a BRCA1 primer set. As native Jurkat
DNA is not methylated at the BRCA1 locus, this primer set is an ideal control foruse in methylation-specific experimentswith Fully Methylated Jurkat DNA.

MBD2b (µg)

1

0

0

1

1

Input

2

3

4

5

6

7

8

Figure 2: Fully Methylated Jurkat DNA is a control for
MethylCollector.
100 ng genomic (lanes 2 & 4) and Fully Methylated Jurkat
DNA (lanes 3 & 5) were assayed with (lanes 4 & 5) orwithout (lanes 2 & 3) inclusion of the MethylCollector
His-tagged MBD2b protein. These samples and input
genomic (lane 7) and Fully Methylated Jurkat DNA (lane8) were then amplified by PCR with the BRCA1 primers.
A positive result is observed only with Fully Methylated
Jurkat DNA captured with the His-tagged MBD2b in the
MethylCollector assay. Lane 8 is a water-only control.

Figure 1: Flow chart of the MethylCollector process.

Product

FormatCatalog No.

MethylCollector™

25 rxns

55002

Fully Methylated Jurkat DNA

10 µg

55003

Toll Free — 1 877 222 9543

April 2008 • volume 9 • number 1

Chromatin Immunoprecipitation

Upgrade your Chromatin IP Experiments using ChIP-IT™ Kits and Reagents
Active Motif’s line of ChIP-IT™ Kits and reagents are a complete, flexible set of tools that make chromatin
immunoprecipitation (ChIP) faster and more consistent. ChIP-IT Kits combine all of the critical components
needed in a single kit, validated and proven to work in ChIP. Kits are available in either sonication or enzymatic
shearing formats, with or without positive and negative controls.
Chromatin IP: The Nexus of Genomics
and Proteomics
Chromatin immunoprecipitation involves
enrichment of protein/DNA complexes
using an antibody to a chromatinassociated protein. It is a versatiletechnique combining the specificity of
immunoprecipitation, the sensitivity of
PCR and the screening power of array
profiling. However, ChIP can be technically challenging and difficult to validate
without well-proven reagents. By providing proven ChIP-validated antibodies,
reagents and controls, you will realize
the best results possible.
Why use ChIP-IT?
• Complete solution – all reagentsand controls supplied• Direct measurement of protein/
DNA interactions• Choice of enzymatic or sonication
shearing methods• No messy phenol/chloroform
extractions• Compatible with genome-wide
profiling or gene-specific PCR-based
approaches• No need to optimize reagents or
protocols
ChIP-IT Control Kits
ChIP-IT Control Kits provide positive &negative control antibodies and speciesspecific PCR primers, PCR buffer and aconvenient DNA loading dye that makes
your PCR reactions ready for loadingon an agarose gel straight from thethermocycler.

www.activemotif.com

ChIP-IT Express: High-throughput ChIP
is now a reality
Active Motif’s innovative ChIP-IT Express
Kits use protein G-coated magnetic
beads, making it possible to simplify
the ChIP protocol, optimize buffers andeliminate several time- and labor-intensive steps. The magnetic beads havemuch lower background than traditional
agarose beads, so pre-clearing and blocking are no longer necessary. Washing iseasier because the spin steps have been
replaced by rapid magnetic pull-down.
Additionally, our specialized ChIP Elution
Buffer eliminates the need for DNA
purification after the ChIP is complete.
The result is that ChIP-IT Express makes
ChIP more successful, more consistentand reduces your time and effort.
ChIP-IT Enzymatic Kits for simplified
chromatin shearing
For successful ChIP experiments, chromatin must first be sheared to 200-1000
bp fragments. Traditionally, shearing is
performed by subjecting chromatin todifferent pulses of sonication, which canbe difficult to optimize and reproduce.
To eliminate the problems associatedwith sonication, Active Motif has developed an enzymatic shearing method toquickly and easily shear chromatin into
200-1000 bp fragments (Figure 1), proving your ChIP results. These enzymatic
shearing components are included in the
ChIP-IT Enzymatic Kits (with or without controls) and the ChIP-IT Express
Enzymatic Kit. They are also available
separately as the Enzymatic Shearing Kit.

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

Ready-to-ChIP HeLa Chromatin
For your convenience, Active Motif
offers Ready-to-ChIP HeLa Chromatin.
Ready-to-ChIP HeLa Chromatin has been
optimally sheared by sonication andvalidated in ChIP. As a result, you canmore easily validate your own antibodies and primer sets. It can be used inconjunction with all of the ChIP-IT Kits
and controls, so you can be certain the
only variable in testing an antibody for
ChIP is the antibody itself.
To upgrade your ChIP experiments, visit
www.activemotif.com/chipit to learn
more about the ChIP-IT line of kits,
reagents and ChIP-validated antibodies.

5Histone Modifications

NEW: High-quality Antibodies to Histones and Histone Modifications toFurther your Chromatin Research
As part of the recent merger with Lake Placid Biologicals, Active Motif now offers high quality, well characterized
antibodies to histones and histone modifications to facilitate your research into histones and chromatin biology.
We have years of experience making antibodies to histone modifications and have developed methods to ensurethat only the best possible antibodies reach your hands. We also work closely with researchers in the field to
produce novel, cutting edge tools for your research.
Organization of DNA into chromatin
DNA is organized by its incorporation into chromatin, 147 base pairs of
DNA coiled around an octamer of core
histone proteins to form the basic
subunit of chromatin, the nucleosome.
The core histones are H2A, H2B, H3 andH4, while histone H1 associates withchromatin outside the nucleosome
and regulates higher order chromatin
structure. Specific functional groups(phospho-, methyl-, acetyl-, ubiquityl-)
are added or subtracted from histone
proteins, and these dynamic additionand subtraction events have profoundeffects on the function of chromatin. In
fact, they are crucial to the regulation of
all genome-based activity: transcription,
chromosome packaging, DNA damage
repair, DNA recombination, etc.
Rigorous specificity testing
Our standards for antibodies are just as
high as those of our customers. When
we make an antibody to a histone
modification, it is crucial that the antibody recognize only the modificationof interest, not a related modification
or a modification at a site with a similaramino acid sequence. To this end, we
employ dot blot analysis on every product we produce, and we make these data
available for review on our web site andour product data sheets.
As most histone-modification antibodies
are generated using synthetic peptides,6

we test ours against the immunogen,as well as the unmodified form of thepeptide. This ensures that the signal
you see in your experiments is indeed
the modification we say it is. We also
screen for reactivity against similar sites
and sequences (Figure 1). Only after anantibody has passed the specificity test
do we further characterize it by testing
it in a variety of applications, including:• Western Blot
• Chromatin Immunoprecipitation• Immunofluorescence
These practices not only result in highly
specific, high-quality antibodies, butthey also enable us to virtually eliminate
lot-to-lot variability. This reflects ourphilosophy that it is important to repeat
your experiments, but not because yourreagents failed.

50
10
2
0.4
Figure 1: Dot blot analysis to confirm the specificity ofHistone H2B acetyl Lys120 pAb.
Modified and unmodified peptides were spotted onto
PVDF and probed with anti-acetyl Lys120 Histone H2B(Cat. No. 39119) at a 1:2,000 dilution. The amount of
peptide spotted (in picomoles) is indicated next toeach row. Lane 1: peptide acetylated at lysine 5 of H2B,
Lane 2: unmodified lysine 5 peptide. Lane 3: peptideacetylated at lysine 16 of H2B, Lane 4: unmodified lysine
16 peptide, Lane 5: peptide acetylated at lysine 120 of
H2B, Lane 6: unmodified lysine 120 peptide.

Figure 2: Immunofluorescence with HeLa cells.
HeLa cells stained with Histone H3 dimethyl Lys9 pAb(Catalog No. 39239) at a dilution of 1:1,000. Top: Histone
H3 dimethyl Lys9 pAb. Middle: DAPI. Bottom: merge.

Please browse our complete line ofhistone and histone-modification
antibodies by visiting us online at:www.activemotif.com/histonemods.

Toll Free — 1 877 222 9543

April 2008 • volume 9 • number 1

HDAC Assay Kits

NEW: Sensitive HDAC Assay Kits in Colorimetric and Fluorescent Readouts
Active Motif’s new HDAC Assay Kits are easy and sensitive assays that can be used to determine histone deacetylase activity as well as to screen potential inhibitor compounds in your cell or nuclear extracts, immunoprecipitatesor purified enzymes.

Why not try it today?
Please visit www.activemotif.com orgive us a call to find out more aboutthe HDAC Assay Kits and related HDAC
antibodies.

www.activemotif.com

Absorbance (OD450)

How does it work?
The HDAC Assay Kits utilize a peptide
substrate that contains an acetylated
lysine residue that can be deacetylatedby Class I, II and IV HDAC enzymes. Class
III HDAC enzymes, or the Sirtuins, require
the addition of the NAD+ cofactor in theassay. Once the substrate is deacetylated, the lysine reacts with the Developing
Solution and releases either the chromophore or the fluorophore from thesubstrate to result in either a colorimetric or fluorescent product. The colorimetric product absorbs maximally at
405 nm and the fluorescent product canbe read with an excitation wavelengthof 360 nm and emission wavelength of
460 nm (Figure 1).

Assay for activity or inhibition
With these simple HDAC Assay Kits,
you can easily assay for HDAC activity
or you can screen compounds for theirinhibitory effects. Also, the provided
deacetylated assay standard enables you
to quantify HDAC effects with greater
accuracy.

HDAC Assay Kit advantages• Works with Class I, II and IV HDAC
enzymes• Easy to adapt to work with Class III
or Sirtuin, HDAC enzymes• Use nuclear extracts, immunoprecipitates, column fractions or
purified proteins• Calculate enzymatic activity andscreen inhibitor compounds
1.0

Trichostatin A

0.8

Untreated

0.6
0.4
0.2
0.0

50

25

12.5

6.25

3.12

1.56

0.78

0

HeLa Nuclear Extract (µg)
50000

Trichostatin A
Untreated

Fluorescence Intensity

What’s in the kit?
HDAC (Histone Deacetylase) Assay
Kits are 96-well plate-based assays
for determining HDAC activity levels
in your extracts or purified samples.
HDAC Assay Kits are available in bothcolorimetric and fluorescent formats tosuit your needs. Each HDAC Assay Kit
contains positive control HeLa nuclear
extract, deacetylated HDAC assay
standard, HDAC peptide substrate,trichostatin A as a model inhibitor andall the essential buffers needed for a
full 96-well plate assay.

40000
30000
20000
10000
0

10

5

2.5

1.25

0.625

0.312

0.156

0

HeLa Nuclear Extract (µg)
Figure 1: HDAC Assay Results.
Colorimetric (top) and Fluorescent (bottom) HDAC Assay results. HeLa Nuclear Extracts were assayed at 0 to 50 µg per
well for the colorimetric assay and 0 to 10 µg per well for the fluorescent assay. Untreated extract results are shown with apurple line, and extracts inhibited with 1 mM Trichostatin A are shown with a copper line.

Product

FormatCatalog No.

HDAC Assay Kit (Fluorescent)1 x 96 rxns

56200

HDAC Assay Kit (Colorimetric)

1 x 96 rxns

56210

7

DNA Damage Assay

DNA Damage Assay – an Easy Assay for DNA Damage Signaling and Repair
Active Motif’s DNA Damage Assay is a sensitive, accurate and simple 2-color fluorescent assay of H2AX
phosphorylation, which occurs following double-stranded breaks in DNA. This early marker of DNA damage makes it
easy to determine the level of DNA breaks in cultured cell samples in response to treatment conditions.
Overview of DNA damage
When cells are exposed to ionizing radiation or certain treatment compounds,
double-stranded DNA breaks are
created that initiate a rapid and highly
coordinated series of signaling events.
Phosphorylation of the histone variant
H2AX at serine 139 to form g-H2AX is
one of the earliest of the DNA-damage
signaling and repair events, and is crucial
for the coordination of downstream
signaling and repair activities. The active
phosphorylation of H2AX has beenshown to spread a distance of up to 1 to
2 mbp from the site of a double-strand
break in DNA in mammalian cells, whichmakes phosphorylated H2AX (Ser 139)a useful marker for the level of DNA
breaks in cells. With Active Motif’s
DNA Damage Assay it is easy to screen
compounds and treatment conditions
for their ability to induce DNA breaks,
repair DNA breaks and study the timingof DNA breaks in cultured cells.
How does the assay work?
The DNA Damage Assay is a cell-based
assay conducted in 96-well plates. Each
kit includes all the reagents necessary
for two 96-well plates. Cells are grown
and treated in the plate, then fixed andincubated with the phospho-Histone
H2AX (Ser 139) double-stranded DNA
damage marker antibody. A short incubation with the included Chromeo™ 488
secondary antibody is performed for
detection. The cells are then washed andstained with propidium iodide and readon a fluorescent plate reader.

8

What’s in the kit?
The DNA Damage Assay includes the polyclonal phospho-Histone H2AX (Ser 139)
antibody, Chromeo 488 Goat anti-Rabbit secondary antibody and the positive control
compound etoposide, which can induce >98% DNA damage (Figure 1).

Figure 1: Percent induction of apoptosis in HeLa cells.
Levels of phospho-H2AX measured in untreated (control)and cells treated with etoposide. Averages of quadruplicates are shown.

Propidium iodide is included to provide a total cell count. This simple detection method
utilizing the superior Chromeo 488 fluorescence and propidium iodide stain makes the
kit ideal for both high-throughput and high-content screening in all cells (Figure 2).

Figure 2: IsoCyte™ single cell analysis of phospho-H2AX detected with Chromeo 488 secondary in HeLa cells.
Untreated HeLa cells (left panel) are detected mostly in the propidium iodide channel while etoposide-treated cells areseen in the green (488 nm) channel indicating a high degree of DNA damage.

Specificity for low background
The assay relies on a high-quality rabbit polyclonal phospho-Histone H2AX (Ser 139) antibody that is subsequently detected with the Chromeo 488 Goat anti-Rabbit secondary.
Our Chromeo fluorescent secondary antibodies are highly specific with extremely low
background, so your results are always accurate.
Start screening compounds today!
Screening treatments for the induction of DNA damage and repair has never been easierwith our DNA Damage Assay. For more information about this assay and our other DNA
Damage and Repair assays, please give us a call.
Product

FormatCatalog No.

DNA Damage Assay (Fluorescent)2 x 96 rxns

18030

Histone H2AX phospho Ser139 pAb

200 µg39117

Toll Free — 1 877 222 9543

April 2008 • volume 9 • number 1

Immunofluorescence

NEW: MAX Stain™ Immunofluorescence Products for Better IF Experiments
Active Motif products for immunofluorescence are the result of our experience with standard, confocal and superresolution microscopy and are actively employed in our own research efforts. Whatever your microscopy method
may be, we are certain you will benefit from the time we have spent optimizing and standardizing the protocols
involved and creating these products.
Immunofluorescence experiments
require high-quality reagents
As biological research causes us tofocus on continually smaller and more
complex interactions within the cellularenvironment, fluorescence microscopyusing antibodies to detect specific proteins (indirect immunofluorescence) is
employed to answer questions with regards to protein localization, abundanceand coincident staining. While technical advances in equipment and optics
have helped to improve image quality,
immunofluorescence experiments
attempting to elucidate the different
properties of proteins ultimately depend
upon the quality of the reagents used.
MAX Stain™ Universal
Immunofluorescence System
Active Motif eliminates the guesswork
and challenge from immunofluorescence
experiments with the new MAX Stain
Universal Immunofluorescence System.
It includes all components required toperform high-quality IF experiments anda detailed protocol – just add cells and a
primary antibody directed against yourtarget protein. MAX Stain includes:• Two sterile 6-well plates withcoverslips to grow your cells• MAXblock™ Blocking Medium• MAXbind™ Staining Medium• MAXwash™ Washing Medium• MAXfluor™ Trio Mounting Media Kit• MAXseal™ Coverslip Sealing System• 24 MAX Stain slides• Chromeo™ 488 anti-rabbit
secondary antibody

www.activemotif.com

MAXblock™ – an highly effective
blocking reagent for immunodetection
experiments
MAXblock is a protein-based nonmammalian blocking agent for
immunofluorescence and immunodetection assays. Superior blocking isachieved utilizing a protein blend that
demonstrates no cross-reactivity withprimary or secondary antibodies (Figure 1).
MAXblock is utilized in all Active Motif
immunofluorescence experiments,
enabling us to provide the best possible
testing data.

Figure 1: Negative controls pushed to their limits.
Methanol-fixed cells were incubated with the indicatedblocking agent, then with a fluorescent secondary antibody (pseudo-colored red) at twice the normal dilution.
Any red observed in the figures represents non-specificbinding of the secondary due to incomplete blocking.
Note that no staining is observed in the MAXblock
panel, demonstrating its effectiveness.

Improve your IF experiments today
Visit www.activemotif.com/maxstain tosee all the IF kits and reagents that will
make your IF experiments easier andmore reproducible.

A

B

C

D

Figure 2: Immunofluorescence performed using the MAX Stain Universal Immunofluorescence System.
Image A: alpha Tubulin mAb (Cat. No. 39527). Image B: Histone H3 acetyl Lys23 pAb (Cat. No. 39131).
Image C: DAPI staining using MAXfluor™ DAPI Mounting Media. Image D: Merge of all three channels.

Product

FormatCatalog No.

MAX Stain™ Universal Immunofluorescence System

1 kit

15250

MAXblock™ Blocking Medium

150 ml

15252

MAXbind™ Staining Medium

250 ml

15253

MAXwash™ Washing Medium

1000 ml

15254

MAXpack™ Immunostaining Media Kit (incl. 15252, 15253, & 15254)

1 kit

15251

MAXfluor™ Mounting Medium

2 ml

15256

MAXfluor™ DAPI Mounting Medium

2 ml

15257

MAXfluor™ PI Mounting Medium

2 ml

15258

MAXfluor™ Trio Mounting Media Kit (incl. 15256, 15257, & 15258)

1 kit

15255

9

TransAM Transcription Factor Assays

Most Published Transcription Factor Assay
TransAM™ Kits are DNA-binding ELISAs that have quickly become the assay of choice for transcription factor
researchers. The combination of a fast, user-friendly format with high sensitivity and specificity makes it easy to see
why over 640 citations are available for TransAM Kits (Figure 1).
Why TransAM is best
The DNA-binding function of activatedtranscription factors is often studied
using EMSAs/gelshifts. But, EMSAs aretime consuming and, at best, provide
only semi-quantitative results. Moreover, they are low throughput, requireradioactive probes and tend to lacksensitivity and reproducibility (Table 1).
TransAM Kits, however, use a uniqueplate-based format to capture activatedtranscription factors, which are analyzed
using antibody specific to your isoform
of interest. TransAM Kits are also nonradioactive and offer up to 100-foldmore sensitivity than traditional gelshifttechniques (Figure 2). This enables you
to detect even the smallest changes intranscription factor levels.

Table 1: Common Methods and Their Properties
MethodNon-Radioactive

Sample Versatility

Functional Assay

Quantitative

Convenience

Western Blot

+
+/+

+
+
+

+
+
+

+/+

+

EMSA
Reporter Assay
TransAM

A complete solution
TransAM’s quantitative and flexible format has made it an invaluable tool for transcription factor researchers. Furthermore, its sensitivity has been proven with various celllines and primary material such as tissues and peripheral blood mononuclear cells(PBMCs). Researchers have used the kits to screen drugs, monitor transcriptional activity regulation, study protein structure/function relationships and more. Consequently,
TransAM is the most widely cited DNA-binding ELISA available (Figure 1). So, why not visit
www.activemotif.com/transam or give us a call to find out more?
A.

IL-1a stimulated
0.5

Widest selection of targets and formats
Not only is TransAM the most citedtranscription factor ELISA, but Active
Motif is proud to offer the broadest
selection of targets, including kits for
NFkB, AP-1, STAT, PPARg, HIF-1, NFATc1,
C/EBP and many others. TransAM NFkB
Kits are also available in a colorimetricor ultra-sensitive chemiluminescentreadout. And, for the ultimate in flexibility, TransAM Flexi NFkB Kits let you
use any capture oligo you choose. Please
visit our website for a complete productlisting, and be sure to check for upcoming new releases!

5

10

25

50

0.5

1

5

10

25

2
5
10 25
Cell extract/well (µg)

5050µg of cell extract

NFkB activation (OD450 nm)

B.
2.5

Unstimulated
IL-1a stimulated

2
1.5
1
0.5
0

Figure 1: TransAM Kit citations.
Using HighWire Press, http://highwire.stanford.edu,
a comparison was made by searching for citations ofTransAM from Active Motif versus the tradenames of allcompetitor kits combined. TransAM is clearly the leader.

101Unstimulated

0.5

1

Figure 2: TransAM NFkB is more sensitive than gelshift.
Human fibroblast WI-38 cells are stimulated with IL-1afor 30 minutes. Increasing amounts of whole-cell extract
are assayed using the TransAM NFkB p50 Kit (B) or gel
retardation (A).

Toll Free — 1 877 222 9543

April 2008 • volume 9 • number 1

Histone Purification

Isolate Pure, Separate Fractions of Histone H2A/H2B and H3/H4 While
Preserving Post-translational Modifications
Now you can easily purify histones and further separate the fractions of core histones from any cell culture or tissue
sample while maintaining post-translational modifications like acetylation, methylation and phosphorylation states.
Histone purification made easy
Active Motif’s Histone Purification Kit
enables you to isolate core histones
from any cell culture or tissue sample(Figure 1). The core histones may be purified as one total population containing
H2A, H2B, H3 and H4, or fractionatedinto separate pools of H2A/H2B dimers
and H3/H4 tetramers (Figure 2).
MW

Log.

Mitotic
ArrestedMW

SodiumButyrateCore Histones

98
64

98

50

64
50

36

36

30

H3
H2B
H2A
H4

16

30
H3
H2A/H2B

16

H4

6

6

4

Figure 1: Core histones isolated from cells and tissue.
Total core histones were isolated from HeLa cells (left)
and rat brain tissue (right).
MW
98
64

investigating the role ofhistones in transcription
or chromatin biology. Our
proprietary buffer systemis optimized to maintainthese modifications while
separating the histones
into pure fractions (Figure 3).

Acid Extraction Method
SodiumButyrate

Mitotic
Arrested

Log.

Active Motif Purification Method
SodiumButyrate

Mitotic
Arrested

Log.
Acetylation

Acid Extraction Method
Log.

Mitotic
Arrested

SodiumButyrate

Active Motif Purification Method
Log.

Mitotic
Arrested

SodiumButyrate

Methylation

How does it work?
Unlike histone purificationby acid precipitation, ourmethod utilizes a uniquepurification resin and a
Figure 3: Post-translational modifications preserved.
series of proprietary elution Acetylation, methylation and phosphorylation states are preserved as well or
better with the Histone Purification Kit vs. a straight acid precipitation method.
buffers to isolate very purehistone fractions. The resin
has a high binding capacity for histones,
Assembly Kit (Catalog No. 53500) to
so core histones can be isolated fromenable the generation of chromatin that
small cell culture samples or from grams
very closely resembles native chromatinof tissue. Sequential elution steps let
for functional assays (Figure 4).
you collect the histones in either one
step containing all core histones or into
two separate populations: one enrichedfor H2A and H2B and a second fraction
containing >90% pure H3 and H4.

50
36
30

16

H2B
H2A

H3
H4

What’s in the kit?
Each kit contains all the necessary equilibration, neutralization and elution buffers plus a reusable purification column
for 10 histone purifications.

1

2

Figure 4: Chromatin assembled with purified histones.
Histones were purified from HeLa cells and used in
the Chromatin Assembly Kit. The ordered spacing of
nucleosomes was confirmed and analyzed by agarose gel.

6
4

Figure 2: Separate H2A/H2B and H3/H4 fractions.
H2A/H2B and H3/H4 fractions isolated from HeLa cells.

Preserve important modifications
Preserving post-translational modifications like acetylation, methylationand phosphorylation is critical when

www.activemotif.com

Better substrate for downstream assays
Core histones isolated by the Histone
Purification Kit method are highly pureand suitable as substrates for downstream assays. Purified histones can beused with the Active Motif’s Chromatin

Try it today!
Call or visit us at www.activemotif.com
to find out more about the Histone
Purification Kit and related products thatwill benefit your research.

Product

FormatCatalog No.

Histone Purification Kit

10 rxns

40025

11

SUMOylation & DNA Methylation

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

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

MW

1

2

3

1

2

3

98
64
50
36
30
16
p53 antibody

SUMO-1 antibody

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

Product

FormatCatalog No.

SUMOlink™ SUMO-1 Kit

20 rxns

40120

SUMOlink™ SUMO-2/3 Kit

20 rxns

40220

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

12Advantages• Flexible – use high G/C content
sequences and uncut DNA• Reproducible – 99% conversion
efficiency• Efficient – reagents and protocol
with proven controls• Easy – DNA purification columns
mean no more precipitations
Try MethylDetector today
MethylDetector will speed and simplify
your methylation analysis. Learn more atwww.activemotif.com.

M

1

2

3

4

5

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

Product

FormatCatalog No.

MethylDetector™

50 rxns

55001

Toll Free — 1 877 222 9543