Immunoprecipitation

samples are on ice/in cold

• Scrape cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see RIPA Buffer).
• For each well in a 12 well plate lyse in 1 ml.
• combine 0.5 ml lysate, 2 ul of antibody and 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).
• rotate 1 hour-overnight in cold room
• spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads
• wash X3: add 1 ml lysis buffer, mix by inverting tube, spin and aspirate supernatent as before.
• spin once more and aspirate all supernatant.
• add 50 ul of sample buffer, boil 5 minutes
• to get rid of beads - open cap, prick bottom with 25G needle 300px (up to halfway of bevel).