Immunoprecipitation with Agarose-Immobilized Antibody
See the protocol for immunoprecipitation with agarose-immobilized antibody, including the reagents needed and procedure outline.
Materials
Cell Lysis Buffer
| NaCl | 7.31 g |
| 1M Tris, pH 8 | 25ml |
| 0.5M EDTA, pH8 | 5 ml |
| 10%NP-40 | 25 ml |
| Distilled H2O | 445 ml |
Store at 4 °C.
4X Sample Buffer
| Glycerol | 4.0 g |
| Tris Base | 0.68 g |
| Tris HCL | 0.67 g |
| LDS | 0.80 g |
| EDTA | 6 mg |
| Brilliant Blue G250 | 2.5 mg |
| Phenol red | 2.5 mg |
Adjust volume to 10 ml with ultra pure water.
Store at 4° C.
4X sample buffer is available from Invitrogen (Cat# NP0007)
1X Sample Buffer
| 4X sample buffer | 150 mcl |
| 1M DTT | 60 mcl |
| Distilled water | 390 mcl |
Make fresh for each use.
Procedure
- Place 500 mcl of the pooled cell lysate (1-3 mg/ml) into a 1.5 ml micro-centrifuge tube.
- To this tube add 15-25 mcl of gel slurry of the Agarose-immobilized Antibody. (For best results, optimal amounts of lysate and slurry should be empirically defined.)
- Rotate the immunoprecipitation reactions (end-to-end) for 3 hours at room temperature or overnight at 4 C.
- Centrifuge (200 x g; 5 minutes) to pellet the complex.
- Remove the supernatant and add 500 mcl cold cell lysis buffer. Centrifuge (200 x g; 5 minutes).
- Repeat wash step 6 twice more. After each centrifugation remove as much of the supernatant as possible.
- After removing the supernatant from the third wash, add 40 mcl of freshly prepared 1X sample buffer to each tube and heat at 90 °C for 5 minutes.
- Load 8 to 16 mcl (20 to 40% of the IP reaction) to a polyacrylamide gel.
Note: For optimal results, complete reduction of the sample is required. We recommend the use of 0.1 M DTT in SDS-PAGE sample buffer and immediately heating samples, loading and running gels. We use 4X sample buffer from Invitrogen (cat#NP0007) to which DTT is added.
