Difference between revisions of "Immunoprecipitation"
From Bridges Lab Protocols
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==Protocol== | ==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. | * 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 27G needle (up to halfway of bevel). can | ||
Revision as of 15:26, 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 27G needle (up to halfway of bevel). can
