Low ChIP Signal from Insufficient Starting Material or Antibody (ChIP (Low Signal))

Question

Accepted Answer

Weak or barely detectable signal across all samples including positive controls. Signal intensity is uniformly low rather than selectively absent at specific regions. Common causes: 1) Starting chromatin amount below 25 μg per immunoprecipitation 2) Antibody concentration below 3 μg per IP reaction 3) Insufficient antibody-to-chromatin ratio for effective capture 4) Low cell number during initial harvest and fixation Solutions: 1) Use 25 μg of chromatin per immunoprecipitation as standard 2) Use 3-5 μg of antibody in first instance for each IP 3) If no signal observed, increase antibody amount up to 10 μg 4) Scale up cell culture to ensure sufficient starting material