Difference between revisions of "Immunoprecipitation"

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* spin once more and aspirate all supernatant.
 
* spin once more and aspirate all supernatant.
 
* add 50 ul of sample buffer, boil 5 minutes
 
* 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
+
* to get rid of beads - open cap, prick bottom with 25G needle [[File:Needle_in_eppendorf_2.jpg|300px]] (up to halfway of bevel).

Revision as of 15:28, 5 April 2012

Protocol

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).