Immunoprecipitation: Difference between revisions

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 ==Protocol==
-* Lyse cells in lysis buffer, such as RIPA buffer with protease inhibitors and phosphatase inhibitors (see [[RIPA Buffer]]).
+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.
-* rotate 0.5 ml lysate with 25 ul of protein A/G plus agarose beads (catalog # sc 2003 santa cruz).
+* 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 27G needle (up to halfway of bevel). can