THE NEWSLETTER OF ACTIVE MOTIF — June 2007 • volume 8 • number 2

Chromeo™
superior fluorescent labels
for improved bioanalysis

2 NEW: Mitotic Assay Kits – Quickly Test Treatment
Effects on Cell Division

IN
THIS
ISSUE

3 Monitoring Phosphorylation of Biomarkers is Simple
and Effective Using In-cell Detection
4 Generate Chromatin In Vitro for Downstream Applications
5 NEW: Histone Acetyltransferase Assay Kit Makes It
Easy to Screen Inhibitors and Test Activity
6 TransAM™: The Most Published Alternative to EMSA
7 NEW: Improved ChIP-IT™ Express – Reduced Background
Improves Results, Enables Less Starting Material

8 NEW: Highly Speciﬁc Isolation of Methylated DNA
9 Antibodies for Epigenetics Research
9 LavaCell™ – a Bright Orange Fluorescent Cell Stain
10 RapidReporter® – Double Destabilization
Improves Response & Sensitivity
11 Simpliﬁed Co-Immunoprecipitation of
Intact Nuclear Complexes
12 NEW: Chromeo™ Fluorescent Secondary
Antibodies

Mitosis Determination

NEW: Mitotic Assay Kits – Quickly Test Treatment Effects on Cell Division
Active Motif’s Mitotic Assay Kits make it simple for you to determine thepercentage of cells undergoing mitosis within a speciﬁc population. The kits
are available in ﬂuorescent, chemiluminescent and colorimetric formats,enabling you to choose whichever assay best suits your research needs.
Cell division is a complex, tightly regulated process that is marked by mitosis.
The Mitotic Assay Kits offer you a simple
and accurate method to determine thepercentage of cells undergoing mitosis
within a speciﬁed population. Because
mitosis is the most deﬁning stage of cell
division, accurate measurement of cells
undergoing mitosis is a valuable method
to characterize the effects of test compounds on cellular progression.
Speciﬁc mitosis marker
Two signiﬁcant mitotic events include
microtubule spindle formation andchromosome condensation. Histone H3
is phosphorylated on serine 28 duringmitotic chromatin condensation before
nuclear division occurs, which makes
phosphorylation of histone H3 (Ser 28)
a reliable marker for cells undergoingmitosis. The Mitotic Assay Kits use ahighly speciﬁc phosphorylated histone
H3 (Ser 28) monoclonal antibody as themitosis marker.
What’s in the kits?
Along with the mitosis marker antibody,
all kits also include paclitaxel to treat
cells for a high-mitotic index reference
population. All kits include a method
for normalizing cell number, and all thenecessary buffers for a simple assay.
Fluorescent kit is ideal for HTS
The ﬂuorescent Mitotic Index Assay Kit
uses a bright ﬂuorescent Chromeo™ 488labeled secondary antibody to detect2

the mitosis marker primary antibody.
Cell number is measured using the propidium iodide included in the kit. This kit
is ideal for use with high-content screening ﬂuorescent scanning technologies,
like the Blueshift IsoCyte™ (Figure 1).

Chemiluminescent and colorimetric
The chemiluminescent and colorimetrickits work similarly to the ﬂuorescent kit
format, but use an HRP-conjugated secondary antibody for detection. Development is then performed with either thecolorimetric or chemiluminescent developing reagents. Paclitaxel is included tocreate a high-mitotic reference population, and cell count can be normalized
by crystal violet staining (Figure 2).

Figure 1: Mitotic Index results on an IsoCyte™ scanner.
Single cell data analysis of HeLa cells treated with 1 µM
paclitaxel for 6 hours.

Figure 2: Mitotic Assay results with colorimetric kit.
Fold induction of mitosis marker in HeLa cells treatedwith 1 µM paclitaxel for 6 hours.

High speciﬁcity and low backgroundThe superior ﬂuorescence detection
with the Chromeo 488 Goat anti-Rat
IgG secondary is highly speciﬁc with extremely low background, so your results
are always accurate (Figure 3).

Start screening today!
Screening the effects of compounds
on cell division is easy with your choiceof ﬂuorescent, chemiluminescent orcolorimetric Mitotic Assay Kits. For more
information, please give us a call.

Figure 3: Mitotic Index results with ﬂuorescent microscopy.
Phospho-Histone H3 (Ser 28) detected with Chromeo 488 Goat anti-Rat IgG is shown in the first image. Nuclear staining of
all cells with propidium iodide is shown in the center image. The merged image on the right show cells stained with bothgreen 488 signal and red propidium iodide signals appearing yellow.

Product

FormatCatalog No.

Mitotic Index Assay Kit (Fluorescent)5 x 96 rxns

18020

Mitotic Assay Kit (Color)

2 x 96 rxns

18021

Mitotic Assay Kit (Chemi)

2 x 96 rxns

18022

Toll Free — 1 877 222 9543

June 2007 • volume 8 • number 2

Phospho-Protein Detection

Monitoring Phosphorylation of Biomarkers is Simple andEffective Using In-cell Detection
Active Motif’s Fast Activated Cell-based ELISA (FACE™) Kits are a simple,
sensitive, cell-based method for detecting protein phosphorylation directlyin the cell, without making cell extracts or the trouble of running gels.
FACE advantages• Cell-based – no extraction, gels, orblotting needed• Accurate – Fixation prevents additional protein modiﬁcations• Fast – 5 hour protocol (only 2 hoursof hands-on time)• Grow cells in 96-well plates – saveon reagents• Economical – Total and phosphospeciﬁc antibody provided for2 x 96 wells• Semi-quantitative results – normalize to total protein and cell number

Add anti-IgG
HRP conjugate

Add primary
antibody
3 hrs.

1 hr.

Cells arefixed following
stimulation

P

P

1

P

Add developing
and stop solution

Stimulatecells
Cells cultured in
96-well plate
Figure 1: Flow chart of the FACE process.
Cells are grown, stimulated and fixed in the same 96-well plate. Addition of primary and secondary antibodies detects
phosphorylated protein.

3

2

4
43 kDa

1

2

3

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

A

4
43 kDaOD450 nmThe “in-cell” method
Fast Activated Cell-based ELISAs (FACE™)
are easy to use and require less than
2 hours of hands-on time. Cells aregrown in 96-well cell culture plates
and treated with a chemical of interest
to change the phosphorylation statusof the protein of interest. After thedesired incubation time the cells are
rapidly ﬁxed with formaldehyde, which
preserves the induced protein modiﬁcations. Each well is then incubatedwith a primary antibody speciﬁc forthe phosphorylation site or the total

protein. Subsequent incubation with asecondary labeled antibody provides acolorimetric or chemiluminescent readout that is quantitative and reproducible(Figure 1). The number of cells in eachwell can be determined easily with theprovided Crystal Violet.

B

Phospho p38
Total p38

C

0

0.1
10
1
Anisomycin induction (µg/ml)

Figure 2: 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

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

FACE™ ATF-2 (T71)

FACE™ Bad (S112)

FACE™ c-Jun (S63)

FACE™ c-Jun (S73)

FACE™ c-Src (Y418)

FACE™ EGFR (Y845)

FACE™ EGFR (Y992)

FACE™ EGFR (Y1173)

FACE™ ErbB-2 (Y877)

FACE™ ErbB-2 (Y1248)

FACE™ ERK1/2 (T202/Y204& T185/Y187)

FACE™ FAK (Y397)

FACE™ FKHR (T24)

FACE™ GSK3β (S9)

FACE™ HSP27 (S82)

FACE™ JAK1 (Y1022/Y1023)

FACE™ JNK (T183/Y185)

FACE™ Maker

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

FACE™ p38 (T180/Y182)

FACE™ PI3 Kinase p85

FACE™ STAT2 (Y689)

FACE™ STAT4 (Y693)

FACE™ STAT6 (Y641)

www.activemotif.com

3Chromatin Assembly

Generate Chromatin In Vitro for Downstream Applications
Active Motif’s Chromatin Assembly Kit enables you to generate chromatin
in vitro from your linear or supercoiled DNA. It yields chromatin that closely
mimics natural in vivo chromatin, so you can discover which histone modiﬁcations and associated proteins are crucial to regulation of your target.
A simple way to generate chromatin
Now you can investigate regulation ofyour gene of interest in its native form
by assembling it into chromatin using
Active Motif’s in vitro Chromatin Assembly Kit. This kit includes all the recombinant proteins, core histones, buffers andATP-utilizing factors needed to generate
chromatin from your DNA sequence, andalso to verify successful assembly. Highquality chromatin with more than six
regularly spaced nucleosomes is made byadding the supplied components to 1 µgof your linear or supercoiled DNA, thenincubating for 4 hours. A simple partial
enzymatic digestion of the resulting
chromatin conﬁrms the ordered spacingof nucleosomes (Figure 1).
M

1

2

3

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 forgene regulation experiments

- +

p53

p300

p53

Figure 2: HAT assay using assembled chromatin.
Chromatin was in vitro assembled with p300 andacetyl-CoA, in the presence (+) or absence (-) of p53 at
30°C for 30 minutes. The sample was then analyzed by
18% SDS-PAGE and visualized following fluorography.

Why bother to make chromatin?
When DNA sequences are assembled
into chromatin by ATP-dependent
factors, the resulting structure closely
resembles the natural chromatin conﬁguration. DNA that is in either a bare or unassembled state often cannot reveal the

Try it today!
The Chromatin Assembly Kit makes it
easy for you to assemble chromatin on
your sequence of interest to investigate
gene regulation in a context that closely
resembles in vivo chromatin. For more
information, please give us a call.

H3
H2B
H2A
H4

1

2

HAT Assay

4

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

Complete kit ensures success
The kit includes recombinant h-NAP-1
chaperone protein, ACF assembly complex, HeLa core histones, and the buffers
and enzymes necessary to generateassembled chromatin from your input
DNA. Control Supercoiled DNA is also
provided. To verify that your chromatin
assembly reaction has been successful,reagents are provided to perform aneasy partial enzymatic digestion to visualize the regular spacing of nucleosomesby agarose gel electrophoresis (Figure 1).

4

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

mechanism of transcriptional activation
or repression with the associated factors
and relevant histone modiﬁcations.
However, properly assembled chromatinwith regularly ordered nucleosomes is an
excellent substrate for subsequent assays such as in vitro transcription assays,
ChIP (Figure 3) and histone acetyltransferase (HAT) assays (Figure 2).

Product

FormatCatalog No.

Chromatin Assembly Kit

10 rxns

53500

Toll Free — 1 877 222 9543

June 2007 • volume 8 • number 2

Histone Acetyltransferase Activity

NEW: Histone Acetyltransferase Assay Kit Makes It Easy to
Screen Inhibitors and Test Activity
Now you can easily assay for histone acetyltransferase activity or screen
potential HAT inhibitors with our simple and sensitive ﬂuorescent histone
acetyltransferase (HAT) assay kit.
An easy assay that works!
High background can make assaying forhistone acetyltransferase activity or inhibition impossible. With Active Motif’s
new Histone Acetyltransferase Assay Kit,
reagents and reaction buffers have been
optimized to assay for activity and toscreen inhibitor compounds.
Fluorescent simplicity
Assaying for histone acetyltransferase
activity is easy with this 96-well plate
format. Simply incubate your HAT withthe histone substrate peptide and acetyl-CoA for 10-30 minutes, then develop!
What’s in the kit?
Active recombinant p300 is provided asa control for use with your samples; it
can also be used as a HAT to screen a fullplate of inhibitor compounds. Histone
H3 and histone H4 substrate peptides
are provided to suit the needs of yourpuriﬁed HAT, and anacardic acid is provided as a potent control inhibitor for
acetyltransferase activity (Figure 1).

How does it work?
The assay utilizes a thiol-reactive ﬂuorescent dye that reacts with the Co-A-SH
generated by the histone acetyltransferase activity to give a ﬂuorescentreadout. The ﬂuorescent dye is excitable
at 360-390 nm and emits at 450-470
nm. This simple reaction chemistry also
makes it easy to generate standard
curves for calculating enzymatic activity.
Determine speciﬁc activity
In addition to screening inhibitors, you
can easily calculate the speciﬁc activityof your histone acetyltransferase. By
preparing a standard curve with acetylCoA or β-mercaptoethanol, you canrelate the ﬂuorescence of your HAT topmol/min/µg speciﬁc activity (Figure 2).

Figure 2: Standard curve to calculate activity.
Acetyl-CoA and β-mercaptoethanol were diluted from0 to 14 µM and used to create a standard curve that canbe used to calculate p300 activity.

Figure 1: p300 activity.
p300 was assayed with 50 µM H3 or H4 peptide with
50 µM acetyl-CoA. Background is shown with p300 andacetyl-CoA. Inhibition of p300 activity was assayed with
15 µM anacardic acid.

www.activemotif.com

Screen for inhibitors
Our assay buffers have been optimized
to ensure there is no interference with
HAT activity or the ability of compounds
to inhibit activity (Figure 3). Dependingon what you want to assay, you can use
the p300 included in the kit to screen afull 96-well plate of inhibitors.
Fluorescent assay advantages• Easy assay• Determine activity of your HAT• Screen compounds

Figure 3: p300 activity and inhibition.
50 ng p300 were assayed per well with 50 µM acetylCoA and 50 µM histone H3 or H4 peptide substrates.
Activity was inhibited with 15 µM anacardic acid and25 µM garcinol.

Try it today!
Call or visit us at www.activemotif.com
to ﬁnd out more about the assay. The
active p300 is also available separately,
so you can use it for other applications. Be sure to visit our website andwatch for additional active histone
acetyltransferases and related assays.

Product

FormatCatalog No.

HAT Assay Kit (Fluorescent)1 x 96 rxns

56100

5 µg

31205

Recombinant p300 protein, catalytic domain

5Transcription Factor ELISAs

TransAM™: The Most Published Alternative to EMSA
TransAM™ Kits are DNA-Binding ELISAs that are quickly becoming 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 are time
consuming and, at best, provide only
semi-quantitative results. Moreover, they
are low throughput, require radioactive
probes and tend to lack sensitivity andreproducibility. TransAM Kits, however,use a unique plate-based format to
capture activated transcription factors, which are analyzed using antibody
speciﬁc to your isoform of interest.
TransAM Kits are also non-radioactive

and offer up to 100-fold more sensitivity than traditional gelshift techniques(Figure 2), which allows you to detecteven the smallest changes in transcription factor levels.
Widest selection of targets and formats
Not only is TransAM the most cited transcription factor ELISA, but Active Motif
is proud to offer the broadest selectionof targets, including kits for NFκB, AP-1,
STAT, PPARγ, HIF-1, NFATc1, C/EBP andmany others (Table 1). TransAM Kits arealso available in either a colorimetric or

ultra-sensitive chemiluminescent* readout. And, for the ultimate in ﬂexibility,
TransAM Flexi NFκB Kits let you use any
capture oligo you choose.
A complete solution
TransAM’s quantitative and ﬂexible
format has made it an invaluable tool fortranscription factor researchers. Furthermore, its sensitivity has been provenwith various cell lines and primary material such as tissues and peripheral bloodmononuclear 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 DNAbinding ELISA available (Figure 1). So, why
not visit www.activemotif.com/transam
or give us a call to ﬁnd out more?

���

2.5
NFκB activation (OD450 nm)

����������������

���
���
���
���
���
���
�

������������

�����������

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.

Unstimulated
IL-1α stimulated

2IL-1α stimulated
0.5

1.5

1

5

10

25

Unstimulated
50

0.5

1

5

10

25

50

1
0.5
0

0.5

1

2
5
10 25
extract/t well (µg)( g)Cell extract/wellµg of cell extract

50

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

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

TransAM™ AML-1/Runx1

TransAM™ C/EBP α/βTransAM™ HNF-1

TransAM™ Flexi NFκB p65

TransAM™ GATA Family

TransAM™ AML-3/Runx2

TransAM™ CREB & pCREB

TransAM™ MEF2

TransAM™ NFκB p65*

TransAM™ HNF Family

TransAM™ AP-1 c-Fos

TransAM™ Elk-1

TransAM™ MyoD

TransAM™ Oct-4

TransAM™ MAPK Family

TransAM™ AP-1 c-Jun

TransAM™ ER

TransAM™ NF-YA

TransAM™ p53

TransAM™ Flexi NFκB Family

TransAM™ AP-1 FosB

TransAM™ FKHR (FOXO1/4)

TransAM™ NFATc1

TransAM™ PPARγTransAM™ NFκB Family

TransAM™ AP-1 JunD

TransAM™ GATA-4

TransAM™ Flexi NFκB p50

TransAM™ Sp1 & Sp1/Sp3

TransAM™ STAT Family

TransAM™ ATF-2

TransAM™ GR

TransAM™ NFκB p50*

TransAM™ STAT3

TransAM™ c-Myc

TransAM™ HIF-1

TransAM™ NFκB p52*

*The TransAM NFκB p50, p52 & p65 Kits are offered in both colorimetric and chemiluminescent formats. TransAM Chemi Kits require the use of a luminometer.

6

Toll Free — 1 877 222 9543

June 2007 • volume 8 • number 2

Chromatin Immunoprecipitation

NEW: Improved ChIP-IT™ Express – Reduced Background
Improves Results, Enables Less Starting Material
Active Motif has improved its ChIP-IT™ Express Kits by greatly reducing thebackground, which improves your results and enables you to use less starting
material than ever before. In addition, the magnetic beads provided with ChIP-IT
Express have made it possible to streamline the protocol so you can get results inhalf the normal time with much less sample manipulation. And, ChIP-IT Express
makes it easy to perform ChIP on many samples at the same time.
ChIP-IT Express advantages• No more need for pre-clearing,
blocking or DNA puriﬁcation steps• Reduced background• High throughput compatible• Dramatically reduced hands-on time
ChIP-IT Express beats the competition
Not only does ChIP-IT Express save you
time, but the simple method generates
better results than traditional ChIP. To
demonstrate, ChIP experiments were
performed using ChIP-IT Express and aleading competitor’s ChIP Kit (Figure 1).

RNA pol II

Neg IgG

RNA pol II

Neg IgG

Competitor ChIP Kit
RNA pol II

Neg IgG

RNA pol II

Neg IgG

ChIP-IT Express

The most efﬁcient ChIP enrichment kit
ChIP is an enrichment technique, nota puriﬁcation method. Thus, the less
efﬁcient your enrichment, the higher
the sample background and the morematerial you will need to obtain aninterpretable result. Conventional ChIP
requires at least 2 million cells as starting
material, which can be problematic withsome cell lines. At the least, growing
this many cells is labor intensive. Active
Motif’s improved ChIP-IT Express Kits,
however, have been optimized to provide superior target gene enrichment,

Figure 1: ChIP-IT Express is more efﬁcient and has less background than the competition.
ChIP-IT Express and standard ChIP (with Protein G agarose beads) were performed in duplicate with
RNA pol II antibody and negative control IgG on HeLa cell DNA from 750,0000 cells. PCR was performed for 36 cycles using GAPDH positive control primers.

Product

FormatCatalog No.

ChIP-IT™ Express

25 rxns

53008

ChIP-IT™ Express Enzymatic

25 rxns

53009

ChIP-IT™ Protein G Magnetic Beads

25 rxns

53014

ChIP-IT™ Control Kit – Human

5 rxns

53010

ChIP-IT™ Control Kit – Mouse

5 rxns

53011

ChIP-IT™ Control Kit – Rat

5 rxns53012

Ready-to-ChIP HeLa Chromatin

10 rxns

53015

www.activemotif.com

resulting in unmatched sensitivity. Using
ChIP-IT Express, it is routine to perform
ChIP on material from as few as 750,000
cells and the kits have even been shown
to work with as few as 12,500 cells!
The magnetic bead advantage
The ChIP-IT Express magnetic beads havemuch less background than standard
agarose beads, and this means pre-clearing and blocking steps are no longer necessary. The magnetic pull-down occurs injust seconds, and re-formulated buffers
allow steps to be combined and DNA
puriﬁcation to be eliminated. ChIP-IT
Express is available in both sonicationand enzymatic shearing formats.
Positive controls ensure success
Because interpreting ChIP data can be
difﬁcult, Active Motif has developed acomplete set of controls to help you understand your results and troubleshootyour assays. To provide you with controlsthat are appropriate for your research,
we removed the human-only controls
from ChIP-IT Express Kits and now offer
human, mouse and rat ChIP-IT Control
Kits separately. These provide positive
and negative control antibodies and species-speciﬁc primers, PCR buffer and aconvenient 10X DNA loading dye so yourPCR reactions are gel-ready. All reagentsare quality control tested and validated
to ensure your ChIP assay is working
properly. In addition, we also offer
convenient Ready-to-ChIP HeLa Chromatin, so you can be certain that the only
variable in validating a new antibody for
ChIP is the antibody itself.
Try the best ChIP kit today
For additional information on the new
and improved ChIP-IT Express Kits, visit
our website at www.activemotif.com.

7

DNA Methylation

NEW: Highly Speciﬁc Isolation of Methylated DNA
Active Motif’s new MethylCollector Kit provides users with a fast and easy protocol for isolating
CpG-methylated DNA from limited amounts of cell or tissue samples. Because it uses a recombinant
Methyl-binding protein (MBD2b) to capture DNA, rather than traditional antibody-based
immunoprecipitations, sensitivity is unmatched and the yield is greatly improved.
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. Readyto-use methylated DNA is then eluted
from the beads. 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.

8����������������������������������
��������������������������������

��������������������
����������������������

�������������������
�����������������������������

����������
����������������������������
�������������

���������������
��������������������������

��������������������������

Figure 1: Flow chart of the MethylCollector process.
In a MethylCollector assay, genomic DNA of interest is sheared by either enzymatic digestion or sonication. The sheared
DNA is then incubated with His-tagged recombinant MBD2b protein, which has a affinity for CpG-methylated DNA. These
protein-DNA complexes are captured with nickel-coated magnetic beads and stringent washes are performed to remove
DNA fragments that have little or no methylation. The methylated DNA is then eluted from the beads and PCR is performed on the resulting supernatant, using primers that are specific to amplify the locus of interest.

Positive control
DNA (A431)
M

1

2

3

4

5

Negative control
DNA (HeLa)
6

M

1

2

3

4

5 6

Figure 2: PCR analysis of isolated CpG-methylated DNA using MethylCollector.
Genomic DNA from A431 (positive control DNA) and HeLa (negative control DNA) cells were enzymatically digested by
Mse I for 2 hours. Increasing amounts of fragmented DNA (from 5, 40 and 100 ng) were then incubated for 1 hour with 1 µg
His-MBD2b recombinant protein in the presence of nickel-coated magnetic beads. After washing, CpG-methylated complexes were eluted. The isolated DNA was then analyzed by 36 cycles of PCR using the kit’s control primers, which amplify
a locus that is not methylated in HeLa but highly methylated in A431. PCR on MethylCollector-enriched A431 DNA generates
robust signals that are proportionate to the amount of DNA starting material (left panel, lanes 4 to 6). No signal is observedwith enriched HeLa DNA (right panel, lanes 4 to 6) or in those samples where His-MBD2b protein was omitted from thebinding reaction (left and right panels, lanes 1 to 3). Taken together, these results indicate that MethylCollector specificallyenriches for methylated DNA fragments, and that this enrichment is due to the presence of the kit’s His-MBD2b protein.

Product

FormatCatalog No.

MethylCollector™

25 rxns

55002

Fully Methylated Jurkat DNA

10 µg

55003

Toll Free — 1 877 222 9543

June 2007 • volume 8 • number 2

Antibodies & Live Cell Staining

Antibodies for Epigenetics Research
Active Motif offers a broad range of antibodies related to DNA methylation and repair. All are suitable for Western blotting
and many are ChIP-validated. For additional information, including technical data sheets, please visit www.activemotif.com.
Product

FormatCatalog No.

Product

FormatCatalog No.

CGBP rabbit pAb

200 µl

39203

Histone H4 rabbit pAb

100 µg

39212

DNMT1 mouse mAb
DNMT2 rabbit pAb

100 µg

39204

INHAT-1/TAF-1α/TAF-1β rabbit pAb

200 µl39202

100 µg

39205

INHAT-2/pp32 rabbit pAb

200 µl39213

DNMT3A mouse mAb

100 µg

39206

MBD1 mouse mAb

100 µg39215

DNMT3B mouse mAb

100 µg

39207

MBD3 rabbit pAb

100 µg

39216

HDAC1 rabbit pAb

100 µg40967

MBD4 rabbit pAb

100 µg

39217

HDAC3 rabbit pAb

100 µg

40968

MeCP2 rabbit pAb

100 µg

39218

HDAC4 rabbit pAb

100 µg40969

MLH-1 mouse mAb

100 µg

39219

HDAC5 rabbit pAb

100 µg40970

MRE11 rabbit pAb

100 µg39220

HDAC6 rabbit pAb

100 µg

40971

Rad17 (phosphorylated) rabbit pAb

100 µg39222

HDAC11 rabbit pAb

200 µl39208

TRF2 goat pAb

100 µg39223

Histone H2A rabbit pAb

100 µg

39209

UBE2N rabbit pAb

100 µg39224

Histone H2B rabbit pAb

100 µg39210

UBE2V1α rabbit pAb

100 µg39225

Histone H3 rabbit pAb

100 µg

39088

UBE2V2 rabbit pAb

100 µg

39226

LavaCell™ – a Bright Orange Fluorescent Cell Stain
LavaCell™ is a unique ﬂuorescent compound that is an ideal stain for live or ﬁxed
cells in any cellular imaging application.
Perfect for cellular imaging
LavaCell is a small, neutral, non-toxic
compound that readily diffuses into live
or ﬁxed cells and spontaneously reacts
to produce bright orange ﬂuorescence.
This naturally ﬂuorescent compound is
excited by a variety of common lasers
and can be multiplexed with a wide
range of common blue, green andyellow-emitting probes (Figure 1).
Ideal ﬂuorescent characteristics
Cells require no pre-treatment orpermeabilization for staining as LavaCell is a neutral molecule that diffuses
across cellular membranes. LavaCell thenrapidly reacts with free amine groups onproteins to produce a bright ﬂuorescentsignal (Figure 2). LavaCell does not stain
nucleic acids nor does it adversely affectwww.activemotif.com

cell growth. LavaCell can be excited with
common laser sources at 405, 488 and532 nm, and has a Stokes shift of over
100 nm for peak emission at 610 nm.
LavaCell staining advantages• Excitable with common laser
sources• Long Stokes shift is great for multiplexing• Non cytotoxic• Fluorescent only when boundmeans no wash steps
Try LavaCell to visualize your cells
Call us for more information and to giveit a try today!

Figure 1: Live mouse macrophage cells were multiplex
stained with LavaCell, calcein AM and Hoescht.

Figure 2: Live HeLa cells stained with LavaCell.

Product

FormatCatalog No.

LavaCell™

200 µg15004

9

Luciferase Reporter Gene Assay

RapidReporter® – Double Destabilization Improves Response & Sensitivity

Stop living in the pastStandard luciferase assays are limited by
the fact that basal activity of the cloned
promoter results in accumulation of boththe luciferase mRNA and protein. The
slow clearance rate of these pre-existing molecules substantially delays anddilutes the measurable response. Thus, alarge proportion of the luciferase measured is derived from transcription thattook place before the test agent waseven added. As a result, in standard assays transient or relatively minor effects
are hidden and kinetics are inaccurate.
Consequently, drugs or treatments thatare disregarded because they have little
or no detectable effect may often elicit
a 10-fold or more change in transcription.1
Half measures will only get you so far
To solve the problem of long half-life
proteins, some vectors include proteindestabilizing elements, which causes
the luciferase to degrade more rapidly.
Destabilizing the protein, however,
only partly addresses the problem, as
clearance rates are also dependent on
the half-life of the mRNA. As long as thereporter mRNA is intact, it continues to
produce new reporter protein.
Double destabilization is the key
To solve the problem, RapidReporter
utilizes vectors that include both protein
AND mRNA destabilizing elements. Suchdouble destabilizing vectors have been
REFERENCES

1. Voon, C. et al. (2004) Nucleic Acids Research 33(3): e27.
2. Almond, A. et al. (2004) Promega Notes 87: 18-22.

10shown to reduce luciferase half-life anadditional 33% compared to vectors with
only protein-destabilizing elements.2
Measure what’s actually happening
The use of double destabilizing vectors
makes RapidReporter more sensitive andresponsive, so you can detect smallerchanges and get more accurate real-timemeasurement than with non-destabilized reporter gene assays, or those thatdestabilize the protein only (Figure 1).
Your choice of stringency
RapidReporter vectors are offered in
two different stringencies. pRR-High ishighly destabilized, while pRR-Low has
fewer elements, which is appropriate
when studying weaker stimulation.

Pre-made vectors & complete kits
In addition to empty RapidReporter
vectors, Active Motif offers vectors that
contain widely studied promoters. All
are available separately or in complete
assay kits, which also include a positive
control vector and buffers & substrate
optimized for Gaussia luciferase. To
learn more about the only reporter assay
with double destabilization, please give
us a call or visit www.activemotif.com.
16

pRR-HighpRR-Low
pGL3
12Fold induction

Active Motif’s patented* RapidReporter® is the only luciferase reporter geneassay that utilizes both mRNA and protein destabilizing elements. This greatly
reduces background, which increases both the magnitude of the responsethat can be measured after stimulation or repression, and the speed in which
the assay can measure changes in transcription (both increases and decreases).

8

4

0
0

1

2

3

4

5

6

Hours after addition of isoproterenol

Figure 1: More accurate kinetics, higher fold induction.
293 cells transiently transfected with pRR-High-CRE,
pRR-Low-CRE and a pGL3 vector containing CRE wereplated onto 96-well plates. Twenty-four hours posttransfection, cells were stimulated with 4 µM isoproterenol, then measured for Gaussia (pRR vectors) andfirefly luciferase activities (pGL3 vector).

Product

FormatCatalog No.

RapidReporter® Gaussia Luciferase Assay

100 rxns
1000 rxns

33001
33002

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

10 µg
100 rxns

33003
33004

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

10 µg
100 rxns

33005
33006

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

10 µg
100 rxns

33007
33008

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

10 µg
100 rxns

33009
33010

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

10 µg
100 rxns

33011
33012

RapidReporter® pRR-High-STAT3 vector
RapidReporter® pRR-High-STAT3 Assay

10 µg
100 rxns

33013
33014

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

Toll Free — 1 877 222 9543

June 2007 • volume 8 • number 2

Co-Immunoprecipitation

Simpliﬁed Co-Immunoprecipitation of Intact Nuclear Complexes
The Nuclear Complex Co-IP Kit simpliﬁes co-immunoprecipitation of intact
nuclear protein complexes because its reagents have been optimized for
preparing nuclear extracts and immunoprecipitating DNA-bound proteins.
Co-Immunoprecipitation (Co-IP) is often
used to study protein/protein interactions. In Co-IP, a ﬁrst antibody is used
to immunoprecipitate a target antigen,which also co-precipitates any bound,interacting proteins. These are then detected by Western blot using antibodies
targeted against the interacting proteins.
However, traditional Co-IP methods are
not optimal for studying DNA-binding
proteins because their complexes arevery fragile, so are frequently disrupted
during extraction. In addition, thesecomplexes can be altered by the salt
and detergent composition of theimmunoprecipitation buffers, which canfurther complicate their analysis.

Perform Co-IP of DNA-bound proteins
To overcome these problems, the Nuclear Complex Co-IP Kit contains extraction
reagents that were designed to help
maintain nuclear protein complexes. The
kit’s extraction process provides a simple,
effective method for isolating intact
protein complexes contained in nuclear
compartments of the cell, speciﬁcallythose previously bound to DNA. Afterisolation of the intact complexes, thesupplied Co-IP reagents were designed
so that you can vary the stringency of
the Co-IP buffers. This improves yourresults by making it easy for you to
study any protein complex, whether its
members are tightly or weakly bound.

“The Nuclear Complex Co-IP Kit improves co-immunoprecipitation of
DNA-binding proteins by providing extraction and immunoprecipitation
components that are optimized to maintain nuclear protein complexes.”
Cell nucleus containing the twointeracting proteins of interest

1

2

3

Nuclear extraction

Add antibody
binding beads

Add antibody directed against
one of the proteins of interest

4
Lane 1. Mol. Weight Marker
Lane 2. Extract
Lane 3. negative IP (no ab)
Lane 4. Co-IP

Western blot analysis of the immunoprecipitated proteinsusing an antibody directed against the second protein of interest

Wash & collect theimmunoprecipitated proteins

Immunoprecipitate theproteins of interest

Figure 1: Flow chart of the Co-Immunoprecipitation process.

Product

FormatCatalog No.

Nuclear Complex Co-IP Kit

50 rxns54001

www.activemotif.com

250
1

2

3

Figure 2: Analysis of p33 in the RNA pol II complex.
HeLa cells were grown to confluence on 100 mm plates
and nuclear extracts were prepared using the kit’s
extraction reagents. For IP experiments, the stringencyof the IP High Buffer was increased by supplementing
with NaCl and Detergent. 100 µg of nuclear extract wasused per IP reaction and incubated with either 2 µg
p33 antibody or no antibody. Following the IP, Western
blot analysis was performed using RNA pol II mouse
mAb at 0.1 µg/ml followed by anti-mouse HRP at 1:1000.
Detection of the p33/RNA pol II complex by the RNA
pol II antibody (lane 3) demonstrates that the Co-IP
was successful in maintaining the protein complex. The
input HeLa extract (lane 1) was run as a control for theWestern blot using 0.1 µg/ml RNA pol II.
Lane 1 Western blot control
Lane 2 Negative Control (no antibody used in IP)
Lane 3 Co-IP: IP using p33/WB using RNA pol II

Easy to optimize for ideal stringency
With the Nuclear Complex Co-IP Kit,
nuclear extracts are prepared using
low-salt buffers and enzymatic shearing.
The low-salt buffers keep the protein
complexes intact, while digestion
gently releases them from the DNA.
Immunoprecipitation is then carriedout to detect the bound proteins. The
kit contains high- and low-stringency IP
buffers, as well as salt and detergent.
Addition of salt and detergent is ideal
for robust protein/protein interactions
because higher stringency reduces background. However, as unstable protein
complexes may not withstand highstringencies, the kit makes it simple tooptimize the stringency as required foreach particular protein complex.
Find the complex members you missed
The Nuclear Complex Co-IP Kit offers
you a simpler, more ﬂexible alternative
than traditional co-immunoprecipitation
methods. To ﬁnd out more about how it
can help you, please give us a call or visit
us on the web at www.activemotif.com.

11

Fluorescent Detection

NEW: Chromeo™ Fluorescent Secondary Antibodies
Now the superior ﬂuorescence properties of the Chromeo™ Dyes areavailable as mouse and rabbit secondary antibody conjugates to provide
you with ﬂexible options for your assays.
New Chromeo 488 nm label
Chromeo Dyes have the superior ﬂuorescent properties you need in an assay,with their bright ﬂuorescent intensity,
stability over a broad pH range, and limited photobleaching. Now, the Chromeo
Dyes are available as 488, 494, 546
and 642 nm conjugates to high-quality
mouse and rabbit secondary antibodies
to suit your ﬂuorescent assay needs.

On the cover:U2OS cells were fixed and permeabilized by methanol.
Tubulin was detected with rabbit primary and Chromeo
488 Goat anti-Rabbit IgG. Cells were also stained withpropidium iodide.

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

Compatible with assay conditions
Chromeo ﬂuorescent secondary antibodies work in all popular ﬁxation conditions such as methanol, formaldehyde
or formalin (Cover image and Figure 1).
Please call or visit us online for more
complete information.

Figure 1: Lamin staining in ﬁxed HeLa cells.
HeLa cells were fixed with formaldehyde and Chromeo
488 Goat anti-Mouse was used to detect a primarymonoclonal mouse lamin A antibody.

Product

FormatCatalog No.

Chromeo™ 488 Goat anti-Mouse IgG

1 mg

15031

Chromeo™ 488 Goat anti-Rabbit IgG=

1 mg

15041

Chromeo™ 494 Goat anti-Rabbit IgG

1 mg

15042

Chromeo™ 546 Goat anti-Mouse IgG

1 mg

15033

Chromeo™ 546 Goat anti-Rabbit IgG

1 mg

15043

Chromeo™ 642 Goat anti-Mouse IgG

1 mg

15034

Chromeo™ 642 Goat anti-Rabbit IgG

1 mg

15044

THE NEWSLETTER OF ACTIVE MOTIF — June 2007 • volume 8 • number 2

PRSRT STD
US POSTAGEPAID
PERMIT #236

92056