EpiCypher has embarked on a massive effort to identify ChIP-capable reagents using Sample Normalization and Antibody Profiling (SNAP-ChIP®), our proprietary recombinant nucleosome spike-in control technology.

Each of our SNAP-ChIP® Certified Antibodies exhibit exquisite in-application performance to deliver reliable ChIP data that you can trust.

Bye Bye Reproducibility Crisis

Name

Cat. No.

Size

Price

H3K4me1

13-0026

100 μg

$399.00

Add to Cart

H3K4me2

13-0027

100 μg

$399.00

Add to Cart

H3K4me3

13-0028

100 μg

$399.00

Add to Cart

H3K9me1

13-0029

100 μg

$399.00

Add to Cart

H3K36me3

13-0031

100 μg

$399.00

Add to Cart

If your PTM of interest is NOT represented in the list, please contact us at info@epicypher.com

Join our mailing list to receive updates on new antibodies. Antibody screening services are also available test your favorite antibody.



SNAP-ChIP® as a tool for antibody validation

SNAP-ChIP® uses DNA-barcoded recombinant designer nucleosomes (dNucs) as next-generation spike-ins for ChIP (Right Figure). This technology enables the identification of high-quality antibodies by rigorously testing antibody performance directly in a ChIP application.

In our recent paper published in Molecular Cell, we applied SNAP-ChIP to directly address the antibody reproducibility crisis.

Conclusion: many commercial 'ChIP-grade' antibodies fail to perform as advertised.

Please contact us for more information about SNAP-ChIP at info@epicypher.com

SNAP-ChIP® panels available HERE

Antibody specificity in peptide arrays has no predictive ability for specificity in ChIP

Histone post-translational modification (PTM) antibodies are commonly tested by histone peptide arrays. While this approach has long been considered the "gold standard" for histone PTM antibody validation, it is biophysically very different from ChIP.

We recently tested the performance of 54 ostensible "ChIP-grade" commercial antibodies to H3K4 methyl states using both peptide array and SNAP-ChIP® (Shah et al. Molecular Cell). SNAP-ChIP® data is shown in the heatmap figure to the right. Using these approaches, we were able to identify highly specific antibodies for each H3K4, H3K9, H3K27, H3K36 and H3K36 methyl states. However, antibody specificity in peptide arrays had no predictive ability for specificity in ChIP. This is rather disquieting since various peptide array formats serve as the gold standard in antibody validation, but likely explains why so many incapable reagents have entered the market. Another key finding of this work is that the most commonly used H3K4me3 antibodies, including those used to generate many ENCODE datasets, are highly cross-reactive to H3K4me2 in SNAP-ChIP® (see heatmap). This means that many genomic areas reported as containing H3K4me3 are actually a result of a contaminating H3K4me2 signal.

Your PTM of interest is NOT represented in the list? please contact us at info@epicypher.com

What is SNAP-ChIP® certified? – high specificity and efficiency

EpiCypher has embarked on a massive effort to identify ChIP-capable reagents that show application-specific specificity and efficient target recovery. After testing over 200 antibodies in SNAP-ChIP® only the highest quality antibodies are recommended for ChIP studies.

EpiCypher SNAP-ChIP® Certified Antibodies are best-in-class reagents, with exquisite target specificity and IP efficiency. The graph to the right shows the specificity / efficiency profile for H3K4me1 antibody.

All SNAP-ChIP® Certified Antibodies have been extensively validated using SNAP-ChIP® and meet the criteria outlined in Table 1.

Table 1. Definition of a "SNAP-ChIP® Certified" Antibody.

Cross-reactivity in ChIP is no longer a problem with the use of SNAP-ChIP® certified antibodies

SNAP-ChIP certified antibodies must exhibit cross-reactivity to related PTMs less than 20%. For example, an antibody to a lysine methylation state (e.g. H3K4me1) is tested against EpiCypher's full SNAP-ChIP® K-MetStat panel containing unmodified, me1, me2, and me3 states at 5 lysine sites: H3K4, H3K9, H3K27, H3K36, and H4K20 (examples shown below), enabling assessment of antibody specificity relative to the most likely sources of cross-reactivity (e.g. mono-, di-, and tri-methyl states) and the most widely studied histone methylation PTMs directly in your ChIP experiment. If even a single species in the panel exhibits cross-reactivity above 20% (Figure below right), we do not recommended the use of these antibodies for ChIP studies.

We can help you to start using SNAP-ChIP as internal control for your experiments, contact us at info@epicypher.com

Below are examples of our competitor's antibodies that fail SNAP-ChIP® testing

Our SNAP-ChIP certification guarantees high specificity - less than 20% cross-reactivity

High IP efficiency results in lower assay variability and improves signal

SNAP-ChIP® certified antibodies must recover at least 5% of the target PTM after immunoprecipitation. Unlike sample chromatin where target PTM abundance is inferred by genomic loci controls (e.g. euchromatin vs. heterochromatin regions), a major advantage of SNAP-ChIP is that the amount of target PTM input can be precisely defined by examining the spike-in barcoded nucleosomes. By quantifying the amount of target remaining after immunoprecipitation relative to what was added in the input, the percent of target recovered (antibody efficiency) can be calculated. Antibodies with higher efficiencies are desirable since they can be expected to recover higher levels of the target PTM in the sample chromatin, resulting in higher signal and more reproducible ChIP data.

Start using SNAP-ChIP to get more reproducible ChIP data, panels available here

Because antibodies with efficiency scores below this 5% threshold recover little of the target input, the resulting data is noisier and more unreliable. This can be observed in the SNAP-ChIP spike-ins, since each nucleosome in the panel is represented by two distinct barcoded DNAs (two technical replicates). In a single ChIP experiment with low efficiency antibodies, the two barcodes show a large discordance in target recovery (as represented by the standard error bars in Figure, right). Although antibodies can exhibit low efficiency but high specificity (Figure, right), such antibodies are still not recommended for ChIP due to technical variability resulting from low overall signal.

Representative examples of low efficiency antibodies

Since PTM are not equimolar in vivo, antibody specificity data must be carefully assessed

PTMs in vivo are present at different amounts. Thus, any cross-reactivity observed in SNAP-ChIP should be taken into context with known abundances of the PTMs studied and on a on a case-by-case basis.

A case study: H3K4me3 is a low abundance PTM with low tolerance for cross-reactivity

  • Quantitative mass spectrometry analysis showed that H3K4me2 is 3-5 fold higher than H3K4me3 depending on the cell type examined (Peach et al., Mol. Cell. Proteomics 2012). Table shows PTM abundance in cells based on published mass spec data.
  • In the case of an H3K4me3 antibody that cross-reacts ~20% with H3K4me2 in SNAP-ChIP (Figure, right), such cross-reactivity will be 3-5 fold higher in sample chromatin, substantially confounding ChIP data.
  • In contrast, a high abundance mark cross-reacting with a lower abundance mark will be of less concern.
  • When reliable information regarding relative PTM abundances is available, EpiCypher considers this information when evaluating the suitability of an antibody for ChIP.

SNAP-ChIP® panels available

EpiCypher has developed a powerful new approach to characterize antibody performance within the context of a ChIP assay. Using this approach, we are dedicated to deliver best-in-class detection reagetns for the epigenetics field.

Below is a list and description of each SNAP-ChIP® available (or coming soon) from EpiCypher.