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Specificity of the antibody in ChIP - confirmed by SNAP-ChIP validation

Specificity of the antibody in ChIP or in ChIP-seq is crucial for reliable results. The specificity is generally demonstrated by Western blot, whether or not after target knockdown, or in the case of antibodies against modified proteins, by dot blot or sometimes peptide array. Recently, we have added an additional level of validation to our histone modification antibodies confirming the specificity in ChIP using SNAP-ChIP panels: K-MetStat and K-AcylStat (Epicypher). These panels consist of a pool of distinctly modified mononucleosomes, each containing a single or multiple histone modifications (either methylations or acetylations) or an unmodified histone. These artificial nucleosomes are spiked into the sheared chromatin before the immunoprecipitation and the recovery of the different nucleosomes is determined by qPCR with primers specific for each nucleosome. In contrast to generally applied methods to determine the specificity of histone antibodies such as dot blot or peptide array in which the off-target reactions of the antibodies are determined in a different context, the SNAP-ChIP panel allows to monitor and to quantify the actual cross reaction of an histone antibody in ChIP.

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    The use of the SNAP-ChIP panels also allows to determine the optimal antibody concentration. Although often ignored, optimization of the antibody concentration can be crucial for highly specific ChIP and ChIP-seq results. Even with a very specific antibody, using a suboptimal concentration could be detrimental for the results. Using too much antibody may result in an increase of the background and thus a decrease in specificity, whereas insufficient antibody might result in a decrease of the recovery and possibly the loss of less enriched regions. It is therefore very important to determine the best compromise between recovery and specificity. To establish this, titration of our antibodies in ChIP is a standard procedure in our antibody validation pipeline. Until recently, the optimal antibody concentration was determined based on the +/- control ratios and the amount of IP’d DNA, sometimes supplemented with bioinformatic analysis of the ChIP-seq results. This method, however, mainly allows to estimate general background and not actual cross reaction. Using the SNAP-ChIP panel, we can now determine the actual effect of antibody concentration on cross reaction with other modifications (Fig. 1).

Figure1. ChIP results obtained with the Diagenode antibody directed against H3K4me3

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    ChIP was performed on human HeLa cells, spiked with the SNAp-ChIP panel, using different amounts of the Diagenode antibody against H3K4me3 (Cat. No. C15410003). The left graph shows the recovery as determined by qPCR, of the promoter of the active GAPDH and EIF4A2 genes, used as positive controls, and the MYOD1 gene and the Sat2 satellite repeat, used as negative controls. The right graph shows the recovery of the H3K4me1, H3K4me2, H3K4me3, H3K9me3, H3K27me3, H3K36me3, H34K20me3 and unmodified nucleosomes. Even though the left graph suggests the antibody is very specific using antibody amounts up to 10 µg, the right graph indicates that using antibody amounts of 1 µg or higher results in cross reaction with the H3K4me2 modification.

Figure 2. SNAP-ChIP validation confirming antibody specificity in ChIP

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    ChIP assays were performed using human HeLa cells, the Diagenode antibody against H4K20me3 (Cat. No. C15410207) and optimized PCR primer pairs for qPCR. ChIP was performed with the iDeal ChIP-seq Kit for Histones (Cat. No. C01010051), using sheared chromatin from 1 million cells. The chromatin was spiked with a panel of in vitro assembled nucleosomes, each containing a specific lysine methylation (SNAP-ChIP K-MetStat Panel, Epicypher). A titration consisting of 0.2, 0.5, 1 and 2 µg of antibody per ChIP experiment was analyzed. IgG (1 µg/IP) was used as a negative IP control. Recovery of the nucleosomes carrying the H4K20me1, H4K20me2, H4K2me3 and the unmodified H4K20 was determined by qPCR. The figure clearly shows the antibody is very specific in ChIP for the H4K20me3 modification.

SNAP-ChIP validation has been added to our antibody validation pipeline. Generally, an antibody is accepted if the recovery of all other nucleosomes is < 20% of the recovery of the specific nucleosome.

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