Difference between revisions of "Immunoprecipitation"
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* scrape cells in lysis buffer, such as [[RIPA Buffer]] with protease inhibitors and phosphatase inhibitors | * 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. | * for each well in a 12 well plate lyse in 1 ml. | ||
| − | * combine 0.5 ml lysate, 2 | + | * combine 0.5 ml lysate, 0.2-0.5 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 | * rotate 1 hour to overnight in cold room | ||
* spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads (can use crushed tip). | * spin 2 minutes at 5000 rpm and aspirate supernatent, carefully avoiding beads (can use crushed tip). | ||
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* after last wash and aspiration, spin once more and aspirate all supernatant. | * after last wash and aspiration, 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 (very important!!!), prick bottom with 25G needle up to halfway of bevel (can use eppendorf tube holder | + | * to get rid of beads - open cap (very important!!!), prick bottom with 25G needle up to halfway of bevel (can use eppendorf tube holder). Place tube on new eppendorf tube, close cap and see drop flowing through the tiny hole. Spin with hinges of both tubes towards center of centrifuge, without centrifuge lid, for 30 seconds at 4600 rpm. |
* load 10ul of IP on gel. | * load 10ul of IP on gel. | ||
* load original lysate - 1% of original IP lysate volume loaded (when using 0.5ml and loading 10/50ul of final IP, this will be 2ul of lysate 1:1 with sample buffer). | * load original lysate - 1% of original IP lysate volume loaded (when using 0.5ml and loading 10/50ul of final IP, this will be 2ul of lysate 1:1 with sample buffer). | ||
| + | |||
| + | [[ Category: Immunoprecipitation ]] | ||
| + | [[ Category: Protein ]] | ||
| + | [[ Category: Protein-Protein Interactions ]] | ||
Latest revision as of 14:23, 21 August 2016
Protocol
Samples are always kept on ice/in cold room/4 celsius centrifuge
- wash cells once with PBS
- 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, 0.2-0.5 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.
- add 50 ul of sample buffer, boil 5 minutes.
- to get rid of beads - open cap (very important!!!), prick bottom with 25G needle up to halfway of bevel (can use eppendorf tube holder). Place tube on new eppendorf tube, close cap and see drop flowing through the tiny hole. Spin with hinges of both tubes towards center of centrifuge, without centrifuge lid, for 30 seconds at 4600 rpm.
- load 10ul of IP on gel.
- load original lysate - 1% of original IP lysate volume loaded (when using 0.5ml and loading 10/50ul of final IP, this will be 2ul of lysate 1:1 with sample buffer).
