SDS-PAGE & Western Blot Protocol (NBP1-54576)

SDS-PAGE & Western Blotting

1) Wash the cells 3 times with PBS and suspend with 10 volumes of cold Lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM 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. Measure the protein concentration of the supernatant and add the cold Lysis buffer to make an 8 mg/mL solution.
3) Mix the sample with an equal volume of Laemmli's sample buffer.
4) Boil the samples for 2 minutes and centrifuge. Load 10 uL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis.
5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for specific transfer procedure.
6) 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.
7) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 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.).
8) Wash the membrane with PBS (5 minutes x 6 times).
9) Incubate the membrane with the HRP-conjugated anti-mouse IgG diluted with 1% skimmed milk for 1 hour at room temperature.
10) Wash the membrane with PBS (5 minutes x 6 times).
11) 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.
12) 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.