Difference between revisions of "Immunoprecipitation"
From Bridges Lab Protocols
| Line 10: | Line 10: | ||
* 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 | + | * 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).
