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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
10%NP-40   25ml
Distilled H2O   445ml

Store at 40C.

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:

  1. Place 500 mcl of the pooled cell lysate (1-3 mg/ml) into a 1.5 ml micro-centrifuge tube.
  2. 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.)
  3. Rotate the immunoprecipitation reactions (end-to-end) for 3 hours at room temperature or overnight at 4 C.
  4. Centrifuge (200 x g; 5 minutes) to pellet the complex.
  5. Remove the supernatant and add 500 mcl cold cell lysis buffer. Centrifuge (200 x g; 5 minutes).
  6. Repeat wash step 6 twice more. After each centrifugation remove as much of the supernatant as possible.
  7. 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.
  8. 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.