Protocols
Immunoprecipitation with Agarose-immobilized Antibody
Reagents Needed:
Agarose-immobilized Antibody
Cell Lysis Buffer
|
NaCl
|
|
7.31g
|
|
1M Tris, pH 8
|
|
25ml
|
|
0.5M EDTA, pH8
|
|
5ml
|
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10%NP-40
|
|
25ml
|
|
Distilled H2O
|
|
445ml
|
Store at 40C.
4X Sample Buffer
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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.
