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The Rockland Advantage: Epigenetic Antibodies – Unsurpassed Expertise
One of the most important biomedical discoveries made over the past ~20 years has been the
identification of “epigenetic modifications” and linking these modifications to the regulation of
a myriad of cellular processes. More recently, many pathological states such as cardiovascular
diseases, addictive disorders, myopathies, metabolic disorders, and numerous cancers are
linked at least in part, to epigenetic modifications.
Rockland’s Epi-PlusTM Epigenetic Antibodies
have been produced with the utmost care at
each step of the manufacturing process.
Starting from the production of peptide
immunogens, assessment of the immune
response throughout the immunization process,
optimization of the purification, and the multiassay quality control of the final antibodies.
Controlling each step in the process results in
the production of reagents that are unsurpassed
in their quality, reproducibility and specificity. So
how does Rockland insure that their Epi-PlusTM
Epigenetic Antibodies are the best in class?
Figure 1. Image of epigenetic marks on DNA as
methylated cytosine bases on each strand.
Courtesy of Christoph Bock (Max Planck Institute ).
1. It All Starts With The Peptides. As with the production of any anti-peptide antibody, the
design of the peptide is critical, as is its quality. In the case of antibodies to epigenetic targets,
the specific region of protein to be used cannot be altered, as it relates to the location of the
epigenetic mark. The quality of the peptide and of the epigenetic building blocks (lysine
monomethyl, dimethyl, etc) is extremely critical to the eventual production of high quality
epigenetic antibodies. This is due to the fact that the epigenetic modification(s) that is/are being
added by the KMTs (lysine methyltransferases) or RMTs (arginine methyltransferases) are
structurally quite similar to each other (see Figure 2 below). From an antibody point of view,
the structures of methylated lysines and arginine differ only slightly from related forms and when
compared to the other antibody determinants that make up the antibody epitope(s), the
difference between a single methyl group and two methyl groups for example is quite small.
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Given this similarity, peptide
immunogens need to be of high
quality in order to generate as
specific an immune response as
possible.
Some manufacturers
utilize amino acid building blocks of
inferior quality, resulting in
contamination of the final peptide
immunogen with other methylated
forms. Due to the manufacturing
process of methylated amino
acids, some supplies of these
building
blocks
can
be
contaminated
with
other
methylated states, and if these are
Figure 2. Structural similarities of methyl modifications added to arginine
utilized for the production of
and lysine residues. Courtesy of Yi Zhang, LCI, UNC, Chapel Hill, NC.
epigenetic peptides for antibody
production, it can result in peptides
that are contaminated with other methylated states. The peptides are used not just to generate
the immune response, but also to demonstrate specificity and in the case of polyclonal antibodies
(pAbs), affinity purification as well as immunodepletion of related, but non-target antibodies.
Simply stated, Rockland utilizes the highest quality peptides possible; if the peptide immunogens
are not high quality the resulting antibodies will not be “best in class.”
2. Proper Quality Control during Antibody Production.
Commercially available
epigenetic antibodies can be either monoclonal (mAb) or polyclonal (pAb) in nature. In general,
the advantages of each type of antibody have been well documented, however for epigenetic
modification detection, there are many reasons why pAbs can be the preferred type of antibody
to utilize.
First, epigenetic antibodies (and antibodies to any post-translational modification) utilize short,
limited peptides to generate the immune response. This will limit the pAb response to only a
short, specific region of a protein and minimizes or even eliminates the issue of specificity when
pAbs are generated using large constructs (expressed protein, isolated native protein) as
immunogens. Second, although short peptides are used to generate the immune response(s),
pAbs have the advantage of producing multiple antibodies to the immunogen, and as such tends
to have a higher affinity for the target region. Third, since a large pool of serum can be collected
from immunized animals, it is possible to generate large amounts of antibody to insure a longterm supply. Fourth, and most importantly, the serum may contain antibodies that recognize
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the target modification but could contain antibodies that recognize other modifications in some
cases. But these can be removed post-serum collection through immunodepletion of the final
antibody. In the case of mAbs, this is not possible, as the clones are specific for a single target
epitope. Why is this a critical distinction? Because unless during the cloning/subcloning process
every possible modification is used during the screening process including those that are not yet
discovered, there is no guarantee that the mAbs that appear to be specific during production
are truly monospecific. Indeed, many mAbs that are commercially available are indicated to
detect not just the target modification, but related targets as well.
Recently, mass spectroscopy (MS) based proteomics has identified epigenetic modifications of
histone lysine residues that do not involve acetylation or methylation. These include formyl,
propionyl, butyryl, succinyl, and malonyl modifications (for review see: Bartke, et al. Brief Funct
Genomics. 2013 May; 12(3): 205–218). To date, there is no published data as to the specificity of
existing antibodies against the myriad of possible modifications; however pAbs have the
advantage
of
possible
post-production
immunodepletion when required. Once a cell line
has been established for the production of a
mAb, that antibody is set in stone. Therefore if
issues with specificity are observed at this point,
the only way to remedy this situation is to go
back to parental cell lines and reclone the desired
antibody (if indeed they are even available) or
begin the antibody production from scratch, with
improved emphasis on screening. A pAb can be
Figure 3. Example of novel histone post translational
immunodepleted if required to provide the
modifications of lysine determined by MS proteomics.
needed specificity when one or more new
modifications are observed.
3. Proper Quality Control of the Final Antibody Product. Across multiple manufacturers,
there is no clear standard as to what constitutes proper quality control of antibodies directed
against epigenetic targets. The most common analyses performed to validate these antibodies
are ELISA, westerns (with and without competition), immunofluorescence, ChIP, and dot blots.
Many antibodies work well in one or more analyses but the lack of specificity is observed in
others. This conundrum has led to the introduction of antibodies with unknown specificity. To
somewhat combat this unknown factor, the modENODE project has published a study of many
epigenetic antibodies (http://compbio.med.harvard.edu/antibodies/). This has helped
tremendously however there are drawbacks to the study. First, this study is now 3 years old,
and many antibodies are not included. Second, their acceptance criteria require just 75% for
dot blot specificity and 50% for western blot analysis of nuclear extracts. Passing ChIP analysis
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required that “…Pearson correlation is more than 0.8 on any pair of ChIPs performed from independent
preparations matched for stage, cell type or biological tissue…” A look through their database
indicates that many antibodies fail to meet one or more of the above criteria and even when
indicated as passing, some antibodies show cross-reactivity with related peptides at levels that
should be deemed as unacceptable (5-10% cross-reactivity with one or more other peptides for
example).
K9-Ac
K9-Me3
K9-Me2
K9-Me1
Non Mod
To overcome these shortcomings, Rockland
insures that each of its antibodies is validated
in multiple types of assays. Dot blots, ChIP,
westerns, and IF are used to assess every antibody.
pM
In cases where there is ambiguity in dot blot data but
100
other data demonstrates specificity, Rockland has
10
gone the extra mile to utilize full length, synthetic H3
1
or H4 containing specific epigenetic modifications at
the site of interest (Epi-SynH3TM or Epi-SynH4TM, 21st
Century Biochemicals, Inc.). The use of these full
pAb 0.5 µg/mL
length histones allowed for a more robust analysis of
Figure 4. Dot blot showing specificity of AntiHistone H3 [Dimethyl Lys9] (RABBIT) Antibody
antibody specificity given that these full length
p/n 600-401-I70. Anti-dimethyl K9 only reacts
histones contain just the modification of interest and
with dimethylated forms of lysine at position 9
westerns can be used in place of dot blots that are
and not mono methyl, tri- methyl or acetylated K9.
based upon short peptides. As an example of our QC,
in figure 4 we show specificity for the modification, the site and the degree of methylation.
4. Antibody Specificity versus Intended Application. When choosing an antibody for
your intended application, the specificity of the antibody versus its sensitivity can be of
paramount importance. In some cases it is necessary to obtain an antibody with the most
specific profile possible however this is not always the case. Take the case of a high throughput
screen utilizing a synthetic peptide substrate where lysine 27 on histone H3 is unmodified and
where the goal is to identify drug candidates that inhibit the monomethylation of this peptide –
H3K27Me0 methylated to K3K27Me1. The required specificity of the antibody used to monitor
this reaction is simply an antibody that is specific for any of the methylated ‘forms’ of H3K27.
Since the screen uses an unmodified peptide, the detection of any methylation – that requires
monomethylation first takes place – if the chosen antibody’s cross-reactivity does NOT include
detection of the unmodified peptide, than this antibody can be used in this screen. This may
allow for the use of an antibody with high sensitivity and thus the need to purchase lesser
amounts of antibody (or obtain a better signal to noise ratio). In many cases antibodies that
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cross-react with related peptides have higher sensitivity – sensitivity that can be reduced in the
case of pAbs if immunodepletion is required to obtain absolute specificity.
In the case of immunostaining, ChIP or other assays where the goal is to detect the activity
associated with one specific modification at a given site, it is far more important to utilize
antibodies that have as little cross-reactivity as possible. It is also critical that western blot
reactivity be limited to just the histone (or other epigenetically modified protein being studied)
and that other proteins are not detected. This is not the case with many of the antibodies
commercially available. If the antibody is being used in a high throughput screen such as
discussed above, cross-reactivity with other proteins is immaterial. However many of these
same cross-reactive antibodies are also used for in situ analyses where other proteins are
present and those studies would be invalid owing to the lack of protein specificity of the antibody
used.
Featured Antibodies for Epigenetics
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H3 Antibodies
H4 Antibodies
HDAC Antibodies
TET Antibodies
Antibody & Assay Development Services
Rockland antibodies for Epigenetics
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Highest quality peptide building blocks
Proper Controls during production
Multi-assay validated
Multiple species validation
Exquisite specificity and sensitivity
Rockland’s quality is undisputed. Compromise elsewhere.
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