THE NEWSLETTER OF ACTIVE MOTIF — September 2009 • volume 10 • number 3

The Impact of DNA Methylation and Chromatin Modiﬁcation on
Development and the Regulation of Gene Expression
2 REVIEW: Epigenetics and Differences
Between Monozygotic Twins andCloned Animals

IN
THIS
ISSUE

4 NEW: HiLite™ Binding Assays – Innovative
Assay uses Fluorescence Polarization to
Measure Binding of Chromatin-modifying
Proteins to Histone Tails
5 RECOMMENDED: Informally Informative
Coverage of Epigenetics
6 NEW: Histone Modiﬁcation ELISAs to
Quantitate Methylation Levels
7 Easily Purify Core Histones from Any Cell
Culture or Tissue Sample

7 NEW: Histones Containing Speciﬁc
Methylated Lysine Residue Modiﬁcations
8 Improve Methyl-CpG Enrichment withMethylCollector™ Ultra
8 Novel UnMethylCollector™ Assay Isolates
Unmethylated CpG Islands
9 NEW: Histone Antibodies with Supplies
that You Can Rely On
9 NEW: Chromatin-associated Enzymes
from Active Motif
10 Simple, Low Background Co-IP of Both
Nuclear & Whole-cell Complexes
11 In-cell Westerns Make it Easy to Monitor
Activation by Phosphorylation

Review Article

Epigenetics and Differences Between Monozygotic Twins and Cloned Animals
Monozygotic twins and cloned animals are considered genetically identical, but signiﬁcant phenotypic discordances
between them may exist. Epigenetic differences between individuals with identical DNA sequences can explain inpart these discordances.
While monozygotic twins and cloned
animals are considered to be genetically
identical, signiﬁcant phenotypic discordances between them may exist. These
differences may in part account fordiscordant expression of disease in suchpairs, including psychiatric diseases (e.g.
schizophrenia, bipolar disease, depression, autism, eating disorder, alcoholism
and other addictions, suicidal behavior
and hyperactivity) and autoimmune
diseases (e.g. juvenile diabetes, multiple
sclerosis, systemic lupus and rheumatoid
arthritis).1-3 Also observed are increasing
degrees of phenotypic discordances
during aging of monozygotic twins orcloned animals.
The causes of incomplete concordance
between individuals with identical DNA
sequences are not clear, and this phenomenon represents one of the mostchallenging questions in complex, nonMendelian diseases. These discordances
can be attributable to environmental exposures,4-6 to the quality of medical andspousal care, and to genetic changes.
Indeed, point mutations and aberrant
chromosomal segregations are known
to have an impact upon the emergenceof neoplastic diseases. Several subtypesof human endogenous retroviruses werealso identiﬁed using representational
difference analysis in monozygotic twins
discordant for schizophrenia, but theirrole in the etiopathogenesis of the disease is not clear.7 Epigenetic differences
may be an important part of the solution to this puzzle. Moreover, epigenetic
proﬁles may represent a link between
an environmental factor and phenotypic
differences between monozygotic twinsor cloned animals.

2

Epigenetics refers to both heritable andtransient changes in gene expressionthat do not entail a change in primary
DNA sequence. These changes include
DNA methylation and post-translational
modiﬁcations of the histone proteins.
Methylation of mammalian DNA haslong been recognized to play a majorrole in different cellular functions, such

A substantial degree of epigenetic
discordance is accumulated overmillions of mitotic cell divisions.
as development and control of gene
expression, and is generally associatedwith transcriptional repression. The DNA
methyltransferases (DNMTs) catalyze
the transfer of a methyl group from
S-adenosyl methionine to the
5´ position of cytosines, mostly within
CpG dinucleotide motifs (reviewed
in 8). Reported histone modiﬁcations
include acetylation, methylation,
phosphorylation, ubiquitylation,
glycosylation, ADP-ribosylation, carbonylation and SUMOylation. Many histoneamino acids are modiﬁed, including
lysine residues (acetylated, methylated
or coupled to ubiquitin), arginine residues (methylated), and serine or threonine residues (phosphorylated). These
modiﬁcations are positively or negatively
correlated with speciﬁc transcriptional
states, or the speciﬁc organization ofrepressive or open chromatin. Many
modiﬁcations affect one another; collectively they constitute the “histone code”.
In this review, we will focus on the
involvement of epigenetics in somediscordances observed in genotypically
identical individuals.

Epigenetic differences during the lifetime of monozygotic twins
Increasing degrees of phenotypic discordances are observed during the lifetimeof monozygotic twins. Fraga et al. haveshown by observing DNA methylationand histone acetylation of a large cohortof monozygotic twins that, althoughtwins are epigenetically indistinguishable
during the early years of life, older twins
exhibit remarkable differences in theiroverall content and genomic distributionof 5-methylcytosine DNA and histone
acetylation, affecting their gene-expression proﬁle.
An interesting example is a reportof twins, both of whom developed
histologically conﬁrmed dementia of
the Alzheimer’s type. But, one had the
diagnosis in her late 60s and the otherwas not diagnosed until age 83.9, 10 It has
also been suggested that epigenetic
dysregulation may make an important
contribution to the etiopathogenesis ofmajor psychoses, which can affect oneof the twins and not the other one.11
Discordant diseases inmonozygotic twins
Beckwith-Wiedemann syndrome (BWS)
A substantial degree of epigenetic
discordance can be accumulated overmillions of mitotic division of cells in genetically identical organisms, due to thepartial stability of epigenetic regulation.
In skin ﬁbroblasts from ﬁve monozygotic twin pairs discordant for BWS, theaffected twins had an imprinting defectat the KCNQ1OT1 gene. The epigenetic
defect is thought to arise from theunequal splitting of the inner cell mass,
which contains the DNA methylation

Toll Free — 1 877 222 9543

September 2009 • volume 10 • number 3
enzymes, during the twinning; thisresults in differential maintenance ofimprinting at KCNQ1OT1. In anotherstudy of twins, mapping of methylated
cytosines revealed numerous subtle
inter-individual epigenetic differences,which are likely to be a genome-wide
phenomenon.12
Systemic Lupus Erythematosus (SLE)
Systemic Lupus Erythematosus is anarchetypal systemic, autoimmune
inﬂammatory disease, characterized by
the production of autoantibodies tomultiple nuclear antigens. Apoptotic
defects and impaired removal of
apoptotic cells contribute to an
overload of autoantigens that initiate
autoimmune response. The highest estimated concordance rate of this diseasein twins is < 60%,13, 14 so the involvementof non-genetic factors in the pathogenesis of SLE must be invoked. The highincidence of twin pairs in which only oneof them has developed SLE supports thenotion that environmental factors andtheir involvement in epigenetic modiﬁcations could have an affect on the
disease. Indeed, as mentioned above,
epigenetic alterations are accumulated
during the individual’s lifetime.15
The epigenetic dysregulation of genes
can contribute to, or increase theactivation of, apoptosis. Moreover,
apoptotic-released material with a
speciﬁc epigenetic pattern may act as an
antigen.16 Epigenetic alterations can also
contribute to an exacerbated activationof T and B cells. T cells from patientswith active lupus were shown to exhibit
globally hypomethylated DNA,17 apparently due to a decrease in the enzymatic
activity of DNA methyltransferases(DNMTs).18 The mechanism by which
hypomethylated T cells induce SLE ispoorly understood. Alterations of thehistone code seem to be implied in thedevelopment of SLE. Indeed, the use ofhistone deacetylase inhibitors suggests

www.activemotif.com

that deacetylation is involved in theskewed expression of certain genes thatare associated with the disease.18
Epigenetic differences between cloned
animals during development
Epigenetic regulation of gene expression is recognized to be one of the
key mechanisms governing embryonicdevelopment and cellular differentiation.
The derivation of the cloned embryosfrom somatic cells, which contain quite
different epigenetic proﬁles compared

Review Article
some is silenced due to DNA methylation and chromatin modiﬁcations. The
process of inactivation is random, which
is why cloning a calico cat will neverproduce the same pattern. During thedevelopment of the cloned cat, thedevelopmental program reactivates both
X chromosomes, and the process ofinactivation recurs in a random manner.
This results in an entirely different coatpattern even when two individuals are
genetically identical.

Although monozygotic twins are epigenetically indistinguishable early of life,
older twins exhibit remarkable differences in their DNA methylation.
to the germline, generates abnormalities of development that can arise frominadequate or inappropriate nuclear
programming.

All these studies show the importanceof epigenetics in the discordances
observed between individuals with an
identical genotype.

The mice agouti gene is responsible forthe coat color of wild-type mice, andisogenic pseudoagouti mice have a rangeof coat colors from yellow to black. The
darkness of the pseudoagouti mice is
proportional to the amount of DNA
methylation in the agouti locus, withcomplete methylation found in black
pseudoagouti mice and reduced methylation in yellow ones.19 The obvious
phenotype of this isogenic mouse strain
is controlled by epigenetic factors. But
these factors are only partially heritable,
which can explain the phenotypic
differences that exist between twocloned mice.

References

The ﬁrst cloned cat was created in Texas
from a calico cat, but was phenotypicallydifferent from her “genetic mother”. Thiscan be partly explained by an epigenetic
phenomenon known as chromosome
X-inactivation. Calico cats are always
female. The gene responsible of the coatcolor is located on the X chromosome.
One allele results in an orange coat color
and another allele of this gene results inblack fur. In female cats, one X chromo-

1. A.J. Silman et al., (1993) Br J Rheumatol 32:903-907.
2. A.G. Cardno, F.V. Rijsdijk, P.C. Sham, R.M. Murray &P. McGuffin, (2002) Am J Psychiatry 159:539-545.
3. A.H. Wong, I.I. Gottesman & A. Petronis, (2005) Hum Mol
Genet 14 Spec No. 1:R11-18.
4. D. Reiss, R. Plomin & E.M. Hetherington, (1991) Am J
Psychiatry 148:283-291.
5. E. Turkheimer & M. Waldron, (2000) Psychol Bull
126:78-108.
6. A. Petronis et al., (2003) Schizophr Bull 29:169-178.
7. P. Deb-Rinker, T.A. Klempan, R.L O’Reilly, E.F. Torrey &S.M. Singh, (1999) Genomics 61:133-144.
8. T. Latham, N. Gilbert & B. Ramsahoye, (2008) Cell Tissue
Res 331:31-55.
9. G.M. Martin, (2005) Proc Natl Acad Sci USA
102:10413-10414.
10. R.H. Cook, S.A. Schneck & D.B. Clark, (1981) Arch Neurol
38:300-301.
11. A. Petronis, (2000) Neuropsychopharmacology 23:1-12.
12. R. Weksberg et al., (2002) Hum Mol Genet 11:1317-1325.
13. D. Deapen et al., (1992) Arthritis Rheum 35:311-318.
14. P. Jarvinen & K. Aho, (1994) Semin Arthritis Rheum
24:19-28 (1994).
15. M.F. Fraga et al., (2005) Proc Natl Acad Sci USA
102:10604-10609.
16. M. Boix-Chornet et al., (2006) J Biol Chem 281:13540-13547.
17. B. Richardson et al., (1990) Arthritis Rheum 33:1665-1673.
18. C. Deng et al., (2001) Arthritis Rheum 44:397-407.
19. H.D Morgan, H.G. Sutherland, D.I. Martin & E. Whitelaw,(1999) Nat Genet 23:314-318.

3

Histone Tail Binding Assay

NEW: HiLite™ Binding Assays – Innovative Assay uses Fluorescence Polarization
to Measure Binding of Chromatin-modifying Proteins to Histone Tails
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 uniqueformat of this assay enables homogenous quantitation of biologically important transcriptional regulation events, andcan be used to determine the speciﬁcity of a particular protein for methylated histone tail peptides.
Identifying proteins that interact with
modiﬁed histones is of great interest
to the research community, as suchproteins are often important regulators of genome function, transducing
the histone modiﬁcation to generate a
speciﬁc cellular outcome. For example,
HP1 and Polycomb proteins have eachbeen shown to bind to histone H3 thathas been methylated at either lysine 9 or
lysine 27. Methylation of these residues
is involved in specifying regions of the
genome that are heterochromatic andtranscriptionally silent, and both HP1 andPolycomb are known to be involved inmaintaining heterochromatic regions.
With Active Motif’s HiLite Histone H3
Methyl-Lys9 / Lys27 Binding Assay, you
can use ﬂuorescence polarization(Figure 1) to determine if your speciﬁc
protein of interest binds histone H3 thatis methylated at either lysine 9 or 27.
You can also measure the afﬁnity of thebinding interactions between yourprotein of interest and speciﬁc histone
methylation states, which in turnenables fast and efﬁcient inhibitor
screening studies (Figure 2).
HiLite Binding Assay advantages• Homogeneous – no washing steps
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

Fluorescent
PeptideDepolarized
Excitation
Light

Polarization
FilterPolarized
ExcitationLight(fast)

Relative
Rate ofRotation

Emitted Lightin Same Plane
as Excitation(slow)

Fluorescent Peptide
bound by Protein
Figure 1: Schematic of the HighLite method that uses ﬂuorescence polarization to measure protein binding.
In HiLite, various fluorescent histone peptides with site-specific modifications are added to wells containing a recombinant
protein (or binding domain), then excited with polarized light. If the peptide is bound by the protein, the rotation of the“bound complex” is much slower than the rotation of unbound peptide. Slower rotation causes the amount of polarized
light that is emitted in the excitation plane to be greater for the complex than for the unbound peptide. This provides aquantitative measure of the histone-binding protein’s affinity for the peptide’s histone modification. With the peptides
included in HiLite, it is possible to determine binding equilibria for proteins that range from 10 kDa to 100 kDa.

Figure 2: Binding of HP1 to histone H3 Methyl-Lys9.
One µM of fluorescent histone H3 Lys 9 peptides, which
are either unmodified or mono-, di- or trimethylated
at K9, were added to a 96-well plate containing a serial
dilution of HP1 protein (x axis) starting at a concentration of 200 µM. The plate was scanned on a Tecan
Infinite F200 using 485 nm excitation and 535 nm
emission filters with polarizers. A gain of 80 was used;
100 reads were taken of each well, then averaged.

What comes in the kit?
HiLite Histone H3 Methyl-Lys9 / Lys27
contains 8 ﬂuorescently labeled peptides
that correspond to the regions ofhistone H3 around lysine 9 and lysine 27
that are either unmodiﬁed, or mono-,
di- or trimethylated. It also contains a
positive control protein, assay buffer,calibration dye and ﬁve 96-well half area
black polystyrene plates. One plate isfor calibrating your microplate reader
and performing a binding curve with thepositive control protein; the remaining
four plates are to assay your protein(s)of interest. For additional details, please
visit us at www.activemotif.com/hilite.

Product

FormatCatalog No.

HiLite™ Histone H3 Methyl-Lys9 / Lys27 Binding Assay

1 kit

57001

Toll Free — 1 877 222 9543

September 2009 • volume 10 • number 3

EpiGenie & Transcription Factor ELISAsRECOMMENDED: Informally Informative Coverage of Epigenetics
EpiGenie is a website dedicated to keeping researchers in touch with the evolving ﬁeld of epigenetics andspotlighting the innovative thinkers who are driving the ﬁeld. It provides highlights on the latest epigenetics
news, emerging methods and new products, as well as interviews with key researchers in the ﬁeld in a casual andlaid-back style that will have you soaking up key happenings effortlessly.
It’s not easy to keep up-to-date with the avalanche of information out there.
That’s why EpiGenie created the most informative site on the Internet (voted
two years in a row; by…them!) dedicated to epigenetics. So, if you’re tired of
not getting the coverage of epigenetics you want, check out EpiGenie’s:• Daily Doses of Epigenetics News with Real-time PubMed Feeds• Weekly Featured Publications You Won’t Want to Miss• Monthly Technical Tidbits in Product Reviews, Featured Products,
and Method Spotlights• Product Database Packed with Epigenetics Research Products• Comprehensive Listings of Epigenetics Conferences, Meetings,
and Symposiums

The EpiGenie website.
EpiGenie features daily news, monthly features, a
database showing kits and antibodies from a wide
variety of companies, and many epigenetic protocols.

So the next time your research grind starts to get the better of you, take a break and spend a few minutes at
EpiGenie. Check them out at www.epigenie.com, or sign up for their Newsletter and they’ll come to you!

Detect Transcription Factor DNA-binding with TransAM™ ELISAs

TransAM advantages• Faster procedure – ELISA-based
assay can be completed in 5 hours,
yet provides more quantitative
results than gelshift assays (Figure 1)• Sensitive – up to 100-fold greater
sensitivity than gelshift assays• Less effort required – our kits arecompatible with multi-channel
pipettors to streamline wash steps• Non-radioactive – colorimetric andchemiluminescent formats available

www.activemotif.com

Flexible ordering options
Active Motif offers a large selectionof TransAM™ Kits for over 40 different
transcription factor targets including
NFκB, AP-1, STAT, and PPARγ to name afew. For a complete product listing andadditional details, please visit us atwww.activemotif.com/transam.
Figure 1: TransAM Kits are more sensitive than gelshift.
Human fibroblast WI-38 cells were stimulated with IL-1α
for 30 minutes. Increasing amounts of whole-cell extract
are assayed using the TransAM NFκB p50 Kit (A) or gel
retardation (B). The TransAM method is 10-fold moresensitive and provides quantitative results.

NFκB activation (OD450 nm)

Looking for a fast and simple alternative to gelshift assays? Active Motif’s TransAM™ Kits are highly sensitive
ELISA-based assays capable of detecting small changes in transcription factor activity without the need for gels orradioactivity. TransAM Kits are 10-fold more sensitive than conventional gelshift assays, while TransAM Chemi Kits
utilize chemiluminescent detection to improve sensitivity up to 100-fold, enabling the use of only 40 ng of extract.
2.5

Unstimulated
IL-1α stimulated

2
1.5
1
0.5
0

A

0.5

1

2
5
10 25
Cell extract/well (µg)

IL-1α stimulated
0.5

B

1

5

10

25

50

Unstimulated
50

0.5

1

5

10

25

50

µg of cell extract

5

Histone Modification ELISAs

NEW: Histone Modiﬁcation ELISAs to Quantitate Methylation Levels
Active Motif has made it easier to screen for changes in histone H3 methylation levels with the introduction of ourHistone Modiﬁcation ELISA kits. We have applied our expertise 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.
Each kit also includes a site- and degree-speciﬁc methylated recombinant histone for use as a quantitative reference.

Histone Modiﬁcation ELISA advantages• Sensitive – works with puriﬁed core
histones and acid extracts• Specific – each modiﬁcation
antibody is tested to ensure low
background and no cross-reactivity
with other site- and degree-speciﬁc
modiﬁcations (Figure 1)• Faster procedure – the convenient
ELISA format saves valuable timeover immunoblotting methods• Controls ensure success – includes
positive control recombinant protein as a reference standard curve
Quantitate methylation levels
Our unique Recombinant Histone H3
proteins with site- and degree-speciﬁcmethylation states are included for use
as a reference standard curve (Figure 2).
A comparison of sample results withknown protein amounts will provide
quantitative data on methylation levels.
Active Motif’s site- and degree-speciﬁcmethylated lysine histones are also available separately (see page 7).

6

How do the Histone ELISAs work?
The Histone Modiﬁcation ELISA kits
include everything necessary to screen
puriﬁed core histones (see page 7), oracid extracts for changes in methylationlevels. A 96-stripwell plate is coatedwith a monoclonal histone H3 antibody
to capture histone H3 from the sample.
Active Motif’s site- and degree-speciﬁc
histone modiﬁcation antibodies areused for detection. An HRP-conjugatedsecondary antibody and developing
solutions provide a sensitive colorimetric readout that is easily quantiﬁed byspectrophotometry.

Histone H3 trimethyl Lys9 (OD 450nm)

Histone H3 Trimethyl Lys9 ELISA
1.8

Recombinant Histone H3

1.6

Recombinant Histone H3monomethyl Lys9
Recombinant Histone H3
dimethyl Lys9
Recombinant Histone H3
trimethyl Lys9

1.4
1.2
1
0.8
0.6
0.4
0.2
0

0

15.63 31.25

62.5

125

250

500

Recombinant protein (ng/well)

1000

Figure 1: Histone H3 trimethyl Lys9 speciﬁcity.
Recombinant Histone H3, mono-, di- and trimethyl
Lys9 proteins were assayed from 15 ng - 1 µg per wellusing the Histone H3 Trimethyl Lys9 ELISA Kit. The
results demonstrate the specificity of the assay. Thereis extremely low background from unmodified histone
H3 and little cross-reactivity for mono- or dimethylated
Lys9. This means that small, specific changes in trimethyl
Lys9 levels can be detected with the ELISA.

Histone H3 Trimethyl Lys27 ELISA
Histone H3 trimethyl Lys27 (OD 450nm)

Histones & methylation
Methylation of lysine residues innucleosomal histones is thought tomediate interactions with the protein complexes involved in regulatingtranscription, replication and DNA repair.
Methylation of lysine 9 and lysine 27 ofhistone H3 has been correlated with heterochromatin formation and transcriptional repression, while methylation oflysine 4 on histone H3 is associated withactive gene transcription in eukaryotes.

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

0

3.125

6.25

12.5

25

50

100

Recombinant Histone H3 trimethyl Lys27 protein(provided positive control)

125

250

5,000

Purified Core
Histones

10,000

HeLa Acid
Extract

Sample (ng/well)Figure 2: Histone H3 Trimethyl Lys27 detection with reference standard curve.
The Histone H3 Trimethyl Lys27 ELISA was used to assay purified HeLa core histones (125-250 ng) and HeLa acid extract(5-10 µg). The provided recombinant Histone H3 trimethyl Lys27 protein was assayed from 3.125-100 ng per well as areference standard curve. The results show the sensitivity of the assay in detecting Histone H3 trimethyl Lys27.

Product

FormatCatalog No.

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

Toll Free — 1 877 222 9543

September 2009 • volume 10 • number 3

Histone Purification & Recombinant Histones

Easily Purify Core Histones from Any Cell Culture or Tissue Sample
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.
Active Motif has two versions of our Histone Puriﬁcation Kit to meet your experimental needs.
How does it work?
Our unique kits use patented methods
with proprietary binding columns andelution buffers to isolate very pure fractions of histones. Using the Histone Puriﬁcation Mini Kit, core histones can be
puriﬁed from as few as 8 x 105 cultured
cells or tissue samples as a single population containing H2A, H2B, H3 and H4.
Core histone isolated with the Mini Kit
can be used in the Histone Modiﬁcation
ELISA Kits to screen for changes in the
methylation level of speciﬁc modiﬁcations of histone H3 (see page 6).

The original Histone Puriﬁcation Kit
enables puriﬁcation of several milligramsof core histones using either a spincolumn or gravity-ﬂow format, eluted as
one total population or fractionated into
separate pools of H2A/H2B dimers andH3/H4 tetramers (Figure 1). Core histones
isolated using the Histone Puriﬁcation
Kit are highly pure and suitable as substrates for downstream assays, including
Active Motif’s Chromatin Assembly Kit,
which generates chromatin in vitro thatvery closely resembles native chromatin
for use in functional assays.

Product

FormatCatalog No.

Histone Puriﬁcation Mini Kit

20 rxns

40026

Histone Puriﬁcation Kit

10 rxns

40025

Chromatin Assembly Kit

10 rxns

53500

Figure 1: Histone Puriﬁcation Kit.
The Histone Purification Kit was used to purify separate
H2A/H2B (Lane 1) and H3/H4 (Lane 2) fractions fromHeLa cells.

NEW: Histones Containing Speciﬁc Methylated Lysine Residue Modiﬁcations
To further investigate the inﬂuence of speciﬁc lysine methylation on the binding of associated chromatin proteins
and transcriptional regulation, Active Motif offers recombinant histones that contain site-speciﬁc mono-, di- andtrimethylated lysines. In addition to our previously released proteins, we now offer methylated H3 Lys36 proteins.
How are they made?
Recombinant histone H3 proteins are
created using a patented approach inwhich an analog of methyl lysine is installed in the histone via a chemical alkylation reaction. This enables the site anddegree of methylation to be carefully
controlled. Each methylation reaction
is over 99% complete and is veriﬁed byhigh-resolution mass spectrometry. The
recombinant histones are also analyzed
by dot blot or immunoblot to conﬁrm
identity. Because the engineered
methylation state closely mimics natural

www.activemotif.com

methylation, these recombinant proteins
are ideal for use in functional assays.

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

Product

FormatCatalog No.

Recombinant Histone H3 (C110A)

50 µg

31207

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

50 µg31208, 31209 or 31210

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

50 µg

31211, 31212 or 31213

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

50 µg31214, 31215 or 31216

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

50 µg31217, 31218 or 31219

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

50 µg

31220, 31221 or 31222

Recombinant Histone H4

50 µg

31223

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

50 µg31224, 31225 or 31226

7

DNA Methylation

Improve Methyl-CpG Enrichment with MethylCollector™ Ultra
The MethylCollector™ Ultra Kit* improves the enrichment of CpG-methylated DNA by incorporating the Methylated
CpG Island Recovery Assay (MIRA), which uses a combination of methyl-binding proteins (MBD2b and MBD3L1) toincrease the afﬁnity for methylated DNA fragments.1 This unique protein complex provides greater speciﬁcity formethylated CpG dinucleotides than antibody immunoprecipitation (MeDIP) methods, and in less than half the time.
“I compared MethylCollector Ultra to a commercial magnetic MeDIP. qPCR analysis
showed that MethylCollector Ultra gave a much better enrichment....it was also aquicker and simpler procedure.” – Irina Bogdarina, Queen Mary University of London
UnMethylCollector™

100

UnboundEluted

80
60
40
20
0

MethylCollector™ Ultra

120
100
ng DNA recovered

120

ng DNA recovered

MethylCollector Ultra advantages• Improved efficiency – high-afﬁnity
binding provides greater enrichment
than antibody immunoprecipitation(MeDIP) methods• Faster procedure – the convenient
magnetic protocol can becompleted in less than 3 hours• Uses minimal sample material –requires as little as 1 ng of DNA(~200 cells), fragmented bysonication or enzymatic digestion• Controls ensure success – includes
positive control DNA andmethylation speciﬁc PCR primers• Versatility – the eluted DNA issuitable for use in endpoint orreal time PCR, bisulﬁte conversion,or ampliﬁcation and labeling for
microarray analysis

UnboundEluted

80
60
40
20

Xist

APC

p48

0

Xist

APC

NY-ESO

Figure 1: Direct comparison of UnMethylCollector and MethylCollector™ Ultra illustrates speciﬁcity.
Both UnMethylCollector and MethylCollector™ Ultra were run according to the protocols using either 200 ng or 100 ng
respectively of the provided Mse I digested human, male genomic DNA. Real time PCR analysis was run across multiple locion both the unbound and eluted fractions. UnMethylCollector clearly captures the unmethylated loci (APC and p48), while
MethylCollector Ultra enriches for the methylated loci (Xist and NY-ESO).

Novel UnMethylCollector™ Assay Isolates Unmethylated CpG Islands
UnMethylCollector™** is the ﬁrst commercially available kit for the speciﬁc isolation and enrichment ofunmethylated CpG islands. The kit utilizes the speciﬁcity of the CXXC binding domain towards unmethylated
CpGs to capture and enrich for DNA fragments that lack methylation.
How does it work?
UnMethylCollector uses a recombinant CXXC protein to speciﬁcally bind
unmethylated DNA fragments containing
as few as one CpG dinucleotide. The kit
provides two buffers: a low-salt buffer
for use with samples containing less than
5 CpGs per fragment and a higher salt
buffer for efﬁcient binding of fragmentswith more than 5 CpGs. Nickel-coatedmagnetic beads capture the protein:DNA
REFERENCE

1. Rauch, T. and Pfeifer, G. (2005) Lab. Investigation 85: 1172-1180.

8complexes, then the unmethylated DNA
is eluted from the beads. Following clean
up, the eluted DNA is ready for use.
A side-by-side comparison of fractions
obtained from both UnMethylCollector

and MethylCollector Ultra Kits illustrates
the speciﬁcity of each technique atbinding and enriching for the appropriate methylation status across multipleloci (Figure 1).

Product

FormatCatalog No.

MethylCollector™ Ultra

30 rxns

55005

UnMethylCollector™

30 rxns

55004

Fully Methylated Jurkat DNA

10 µg

55003

* MethylCollector™ Ultra is covered under U.S. Patent No. 7,425,415.
** Patent Pending

Toll Free — 1 877 222 9543

September 2009 • volume 10 • number 3

Antibodies & Functional Proteins

New: Histone Antibodies with Supplies that You Can Rely On

H3
K2
7m
e1
H3
K2
7m
e2
H3
K2
7m
e3

H3
K9
me
1
H3
K9
me
2
H3
K9
me
3
H3
K2
7

H3
K4
me
1
H3
K4
me
2
H3
K4
me
3
H3
K9

A

H3
K4

We know that having the highest quality antibodies available when you want them is critical to accomplishing yourresearch goals. That’s why Active Motif is proud to be able to produce and test all its own antibodies, unlike manyother companies that “source” their antibodies. This means that we can guarantee our customers not only the quality
they desire, but also the availability. So, if you’re having problems getting hold of important antibodies from yourusual antibody “supplier”, take the time to visit www.activemotif.com/abs today. New antibodies are released every
day, and because we make them ourselves, we’ll have them available tomorrow, or whenever you need to re-order.
BC

Dot blot and Western blot analyses used to verify antibody speciﬁcity to histone modiﬁcations.
A. Decreasing amounts of peptides (picomoles, y-axis) corresponding to unmodified, mono-, di- and trimethylated Histone
H3 Lys4, Lys9 and Lys27 were spotted onto PVDF and probed with Histone H3 dimethyl Lys27 pAb (Cat. No. 39245).
Detection was appreciable only for H3K27me2 peptide (Lane 11), demonstrating the antibody’s specificity. B. Histone H3
trimethyl Lys4 pAb (Cat. No. 39159) tested at a 1:1,000 dilution in Western blot using 10 µg HeLa acid extract (Cat. No. 36200).
C. Histone H4 trimethyl Lys20 pAb (Cat. No. 39180) was tested at a 1:1,000 dilution in Western blot using HeLa acid extract.

New: Chromatin-associated Enzymes from Active Motifwww.activemotif.com

aH
9
Sm 04Kyd
2
Smyd
2
Se Y20
4F
t9
Se
t9
H
Se 297A
t8
Se
t8
D3
38
A

G9

MT
1
G9 E143
Q
a

PR

PR

MT
1

Changes in chromatin structure that result from the posttranslational modiﬁcation of amino acids within the tail of
histones H2A, H2B, H3 and H4 are known to play an important 98
role in regulation of gene expression. As a result, there is a
64
growing scientiﬁc interest in gaining a better understandingof how proteins involved in chromatin modiﬁcation function, 50
and the role they may play in disease processes. One of thesimplest methods for studying protein function and regula- 36
Puriﬁed wild-type and mutated histone methyltransferase proteins.
tion is in a biochemical context. This is why Active Motif
A variety of wild-type and mutated histone methyltransferase proteins wereexpressed in E. coli, purified, run on a gel then Coomassie Blue stained. The
offers an ever-increasing line of functionally active proteinswild-type proteins are active and are for use in methyltransferase assays,
while the mutated proteins have no activity, so are ideal negative controls.
involved in many chromatin-associated processes. Active
Motif has proteins available for the analysis of histone methylation, ubiquitination and acetylation as well as DNA
repair and transcriptional regulation. To ﬁnd out more, please visit our website at www.activemotif.com/proteins.

9Magnetic Co-Immunoprecipitation

Simple, Low Background Co-IP of Both Nuclear & Whole-cell Complexes
The Universal Magnetic Co-IP Kit includes protein G-coated magnetic beads
that simplify the IP and wash steps while providing lower background, which
improves your Co-IP results. 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 ERα.
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, ERα pAb and rabbit IgG (as anegative control). Western blot was then performed
using the ERα pAb on 10 µg Input Extract (Lane 1), SRC-1
IP (Lane 2), ERα IP (Lane 3) and the rabbit IgG IP (Lane 4).

Co-IP cytoplasmic AND nuclear complexes
Co-IP is often used to study cytoplasmic protein complexes. But, traditional
methods are not optimal for studying
DNA-binding proteins because nuclear
complexes are very fragile, causing them
to be disrupted during extraction. For
this reason, in addition to containing
components for preparing whole-cell
extracts, the Universal Magnetic Co-IP
Kit provides nuclear extraction reagentsthat have been optimized to preserve
nuclear protein complexes. The kit’s
Enzymatic Shearing Cocktail uses DNA
digestion to gently release the nuclear
protein complexes from the DNA, sothey are intact and ready for Co-IP.

10Simpler procedure, lower background
The Universal Magnetic Co-IP Kit utilizes
protein G-coated magnetic beads, whichsimplify Co-IP by enabling the IP andwash steps to be performed in seconds,
rather than having to use centrifugation.
Because these beads have very low nonspeciﬁc binding, background is reduced
even while using the kit’s low-salt Co-IP/
Wash Buffer, which is designed to help
maintain weaker 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 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 and protein
modiﬁcations (Figures 2 & 3). Finally, thekit includes a strong bar magnet, so you
can take 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/coip.

Product

FormatCatalog No.

Universal Magnetic Co-IP Kit

25 rxns

54002

Toll Free — 1 877 222 9543

September 2009 • volume 10 • number 3

Phospho-Protein Detection

In-cell Westerns Make it Easy to Monitor Activation by Phosphorylation

FACE advantages• Cell-based – no extraction, gelsor blotting needed• Accurate – ﬁxation prevents
additional protein modiﬁcations• Fast – 5-hour protocol, with just
2 hours of hands-on time
• Simple – grow your cells, stimulate,then assay in the same 96-well plate• Complete – kits include both total
and phospho-speciﬁc antibodies so
you can assay 2 x 96 wells• Semi-quantitative results – normalize to total protein and cell number

treated to induce phosphorylation of
the protein of interest. The cells are
rapidly ﬁxed with formaldehyde topreserve the phosphorylation state,
and then each well is incubated with a
primary antibody speciﬁc for either the
total protein or its phosphorylated form.
Subsequent incubation with a labeledsecondary provides a colorimetric or
chemiluminescent readout that is quantitative and reproducible (Figures 1 & 2).
Data is easily normalized to cell number
using the provided Crystal Violet Dye.

The “in-cell” method
Fast Activated Cell-based ELISAs (FACE™)
are easy to use and require just
2 hours of hands-on time. Cells aregrown in 96-well cell culture plates andA variety of kits to choose from
FACE Kits are available for over 20 different targets (Table 1) in both colorimetricand chemiluminescent formats. The Suspension Cell FACE module was designed
to work with all FACE Kits; it improves
Add anti-IgG
HRP conjugate

Add primary
antibody
3 hrs.

1 hr.

Cells arefixed following
stimulation

P

P

P

results when working with suspension
cells by providing 96-well ﬁlter plates
that make it easier to perform washing &liquid handling steps. With FACE Maker
Kits, you can use your own primary andsecondary antibodies to detect any
target or modiﬁcation state of interest.
Simplify your phospho-assays today!
For more complete information, please
visit us at www.activemotif.com/face.

1

1

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 total
protein as well as the phosphorylated form of the protein.

3

4
A

2

3

4
B

43 kDa

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Add developing
and stop solution

Stimulatecells

2

43 kDaOD450 nmFast Activated Cell-based ELISA (FACE™) Kits are a simple, sensitive, cell-based
method for detecting protein phosphorylation directly in the cell. These
convenient in-cell Westerns eliminate the need to make extracts and run gels.

Phospho p38
Total p38

C

0

0.1
1
10
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 (FOXO1) (T24)

FACE™ HSP27 (S82)

FACE™ JAK1 (Y1022/
Y1023)

FACE™ JNK (T183/Y185)

FACE™ MEK1/2 (S217/S221)

FACE™ NFκB Proﬁler(S468 & S536)

FACE™ p38 (T180/Y182)

FACE™ PI3 Kinase p85

FACE™ STAT2 (Y869)

FACE™ STAT4 (Y693)

FACE™ STAT6 (Y641)

FACE™ Maker

Suspension Cell FACE™

www.activemotif.com

11