ChIP–qPCR Validated Antibodies
Protein-DNA interactions mediate many different epigenetic processes. Researchers studying these interactions need a reliable means of analyzing when and where they occur on the genome. ChIP is widely used to analyze protein-DNA interactions to specific genes and regulatory regions. Coupling ChIP with quantitative PCR (qPCR) provides a powerful tool to generate quantitative, real-time data on protein-DNA interactions within a short turnaround time.
A successful ChIP-qPCR experiment requires antibodies that are sensitive, specific, and provide reproducible results every time. CST rigorously validates the antibodies recommended for ChIP-qPCR to ensure they meet the high quality standards needed for reliable data generation.
ChIP-qPCR Antibody Validation Steps
- Antibody specificity is determined by analyzing immuno-enrichment of at least two (2) known positive and one (1) known negative target loci. The antibody must show a minimum fold enrichment for known positive loci compared to known negative target loci.
- Antibody specificity is further analyzed for histone methyllysine and methylarginine antibodies using CST’s proprietary histone peptide arrays.
- Antibody sensitivity is determined by analyzing the signal:noise ratio of known target loci immuno-enrichment in antibody:isotype control comparisons. The antibody must show a pre-determined minimum signal:noise ratio, that is a minimum acceptable fold enrichment of the target locus by the target-specific antibody over the isotype control.
- Every antibody is titrated to determine optimal performance in the ChIP assay. Too little or too much antibody can both be detrimental to the immune-enrichment of target loci.
- Lot-to-lot reproducibility is confirmed by titrating the new lot vs previous lots.
- Antibody specificity is evaluated using known positive vs negative and wild-type vs knockout cell lines.
- Antibody specificity is evaluated using cell treatments that induce nuclear localization and/or binding to target genes.
- Antibody performance is determined across multiple applications to provide end users with increased confidence in the antibody’s specificity.