Immunoprecipitation with Agarose-Immobilized Antibody
- Agarose-immobilized Antibody
Cell Lysis Buffer
|1M Tris, pH 8||25ml|
|0.5M EDTA, pH8||5 ml|
|Distilled H2O||445 ml|
Store at 4 °C.
4X Sample Buffer
|Tris Base||0.68 g|
|Tris HCL||0.67 g|
|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.
- 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.