High Background from Antigen Degradation During IP (Immunoprecipitation (High Background))

Question

Accepted Answer

High background with smeared or degraded protein bands in IP eluate, often accompanied by loss of target protein signal. Multiple lower molecular weight bands appear below the expected target band. Common causes: 1) Protease inhibitors not added to lysis buffer or have lost activity 2) Protease inhibitors added too late after cell lysis has begun 3) Sample kept at room temperature too long during IP procedure 4) Old or improperly stored protease inhibitor stock solutions used Solutions: 1) Add fresh protease inhibitors immediately when lysing samples, before mechanical disruption 2) Prepare protease inhibitor cocktail fresh or use single-use aliquots stored at -20°C 3) Keep samples on ice throughout the entire IP procedure 4) Complete IP procedure within 4-6 hours and store lysates at -80°C if not used immediately