Background from Insoluble Protein Carryover (Immunoprecipitation (High Background))

Question

Accepted Answer

High background in IP eluate with particulate matter visible, caused by insoluble proteins or cellular debris contaminating the soluble lysate supernatant. Common causes: 1) Supernatant not removed immediately after centrifugation allowing pellet resuspension 2) Insufficient centrifugation speed or time failing to pellet insoluble material 3) Vigorous pipetting or vortexing causing resuspension of pelleted debris 4) Detergent concentration too low to fully solubilize membrane proteins Solutions: 1) Remove supernatant immediately after centrifugation without disturbing the pellet 2) If pellet resuspension occurs, centrifuge again at same or higher speed 3) Use careful pipetting technique, avoiding the pellet region at tube bottom 4) Centrifuge lysate at 12,000-16,000 × g for 10-15 min at 4°C before IP