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* scrape cells in lysis buffer, such as [[RIPA Buffer]] with protease inhibitors and phosphatase inhibitors
* for each well in a 12 well plate lyse in 1 ml.
* combine 0.5 ml lysate, 0.2 ul -0.5 ug of antibody and 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).* for tissue lysates - use 0.5 mg of protein, add primary antibody first for 0.5 hour and then add beads.
* rotate 1 hour to overnight in cold room
* spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads (can use crushed tip).
* wash X3: add 1 ml lysis buffer, mix by inverting tube (no vortex), spin 2 minutes 5000 rpm and aspirate supernatent.
* after last wash and aspiration, spin once more and aspirate all supernatant.