1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl pH 7.2, 250mM NaCl, 0.1% NP-40, 2mM EDTA, 10% glycerol) containing appropriate protease inhibitors. Incubate it at 4C with rotating for 30 minutes, then sonicate briefly (up to 10 seconds).
2) Centrifuge the tube at 12,000 x g for 10 minutes at 4C and transfer the supernatant to another tube.
3) Add primary antibody as suggested in the APPLICATIONS into 100ug of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4C. Add 20uL of 50% protein A agarose beads resuspended in the cold Lysis buffer. Mix well and incubate with gentle agitation for 60 minutes at 4C.
4) Wash the beads 3-5 times with the cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds).
5) Resuspend the beads in 20uL of Laemmli's sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Load 10 uL of the sample per lane in a 1mm thick SDS-polyacrylamide gel for electrophoresis.
6) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25mM Tris, 190mM glycine, 20% MeOH). See the manufacture's manual for specific transfer procedure.
7) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4C.
8) Incubate the membrane with primary antibody diluted with PBS, pH7.2 containing 1% skimmed milk as suggested in the APPLICATIONS for 1 hour at room temperature. (The optimal antibody concentration will depend on the experimental conditions.)
9) Wash the membrane with PBS (5 minutes x 6 times).
10) Incubate the membrane with HRP-conjugated anti-mouse IgG diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature.
11) Wash the membrane with PBS (5 minutes x 6 times).
12) Wipe excess buffer from the membrane, then incubate it with appropriate chemiluminescence reagents for 1 minute. Remove extra reagent from the membrane by dabbing with a paper towel, and seal it in plastic wrap.
13) Expose to X-ray film in a dark room for 5 minutes. Develop the film as usual. The conditions for exposure and development may vary.