ChIP-seq Validated Antibodies

A successful ChIP-seq experiment requires an antibody that recognizes the correct target protein in all sequence contexts across the entire genome. Good antibody performance in ChIP-qPCR does not necessarily mean the antibody will perform well for ChIP-seq, because ChIP-seq requires more extensive capture of the target protein across a large number of gene loci. To provide antibodies that are proven effective for ChIP-seq, CST validates recombinant rabbit monoclonal antibodies for ChIP-seq using the SimpleChIP® Enzymatic and SimpleChIP® Sonication Chromatin IP protocols followed by next-generation sequencing (NGS).

ChIP-seq Antibody Validation Steps

• All ChIP-seq validated antibodies are first subjected to the ChIP-qPCR validation protocol.
• Antibody sensitivity for ChIP-seq is then confirmed by analyzing the signal:noise ratio of target enrichment across the genome in antibody:input control comparisons. The antibody must provide an acceptable minimum number of defined enrichment peaks and a minimum signal:noise threshold compared to input chromatin.
• For sequence-specific DNA-binding transcription factors, antibody specificity is determined by performing motif analysis of enriched chromatin fragments.
• Antibody specificity is further determined by comparing enrichment across the genome using multiple antibodies against distinct target protein epitopes.
• Antibody specificity is confirmed using antibodies against different subunits of a multiprotein complex.
• Antibody specificity is further confirmed by comparing enrichment across the genome to published ChIP-seq data (ie, ENCODE) using additional antibodies for a given target protein.