No Product in Experimental Antibody IP (ChIP (CST Guide))

Question

Accepted Answer

Experimental antibody-IP PCR reaction produces no product while positive control H3-IP works, indicating antibody-specific or target-specific issues. Common causes: 1) Insufficient DNA added to PCR reaction for detection of low-abundance targets 2) Insufficient antibody added to IP reaction (below optimal 1-5 µg range) 3) Antibody not validated or unsuitable for ChIP application 4) Target protein occupancy at genomic locus is lower than expected 5) Antibody epitope destroyed by crosslinking or sonication conditions Solutions: 1) Add more DNA to PCR reaction or increase number of amplification cycles 2) Increase antibody amount (typically 1-5 µg per IP; exact amount depends on individual antibody) 3) Choose an alternate ChIP-validated antibody from reputable vendor 4) Verify target protein binds to selected genomic region using published ChIP-seq data 5) Optimize crosslinking and sonication conditions to preserve antibody epitopes