PREPARATION OF THE CELL LYSATE
- Rinse a 60 mm culture dish of confluent cells with 1X phosphate-buffered saline (PBS).
- Add 0.5 ml boiling lysis buffer (1% SDS, 1.0 mM sodium ortho-vanadate, 10 mM Tris pH 7.4) to the culture dishes.
- Scrape the cells from the dish, transfer lysate to a 1.5 ml microcentrifuge tube, and boil for 5 minutes in a boiling water bath.
- Pass several times through a 26 gauge needle or sonicate to reduce viscosity; centrifuge (16,000 x g) for 15 minutes. The supernatant is the "total cell lysate (denatured)".
- Rinse a 60 mm culture dish of confluent cells with PBS.
- Add 0.5 ml cold immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium ortho-vanadate, protease inhibitor cocktail (Boehringer Mannheim), 0.5% IGEPAL CA-630) to the dishes to lyse cells.
- Maintain constant agitation for 30 minutes at 4°C in order for complete lysis to occur.
- Scrape the cell from the dish and pass several times through a 26 gauge needle to disperse any large aggregates and reduce viscosity. Centrifuge (16,000 x g, 4°C) for 15 minutes; keep on ice. The supernatant is the "total cell lysate (native)".
It is important to pre-clear the lysate immediately before immunoprecipitation.
- To 750-1000 µl of supernatant (denatured lysate or native total lysate), add 5 µg of rabbit anti-mouse IgG antibody, vortex, then add 75-100 µl of Protein A:Agarose (Life Technologies). Incubate at 4°C for 30 minutes with gentle agitation.
- Centrifuge lysate (9000 x g, 4°C) for 2 minutes to pellet the agarose beads. The supernatant is the "total cell lysate".
- To a microcentrifuge tube, add 2–5 µg of anti-PTyr:Biotin, 400 µl of water, 200-500 µg of total lysate in 100 µl, and 500 µl of 2X immunoprecipitation buffer (2% Triton X-100, 300 mM NaCl, 20 mM Tris pH 7.4, 2 mM EDTA, 2 mM EGTA pH 8.0, 0.4 mM sodium ortho-vanadate, protease inhibitor cocktail (Boehringer Mannheim), 1.0% IGEPAL CA-630).
- Vortex and incubate for one hour with agitation at 4°C.
- Add 20 µl of Streptavidin immobilized on agarose (Pierce). Vortex and incubate for 30 minutes with agitation at 4°C.
- Wash with cold 1X immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0.2 mM sodium ortho-vanadate, protease inhibitor cocktail, 0.5% IGEPAL CA-630) by centrifuging 2 minutes (8000 x g, 4 degrees). Decant supernatant and repeat wash twice.
- Resuspend pellet in 50 µl of 0.1 M Glycine pH 2.5 vortex and incubate with agitation for 10 minutes at 4°C.
- Centrifuge (9000 x g, 4°C) for 2 minutes. Remove supernatant, this is your IP sample.
- Add 5 µl of 1 M Tris pH 8.0 to each tube to neutralize the pH. Add approximately 10 µl of 5X concentrated electrophoresis sample buffer (125 mM Tris pH 6.8, 4% SDS, 10% glycerol, 0.006% bromophenol blue, 2% ß-mercaptoethanol) to each sample, and boil for 5 minutes. Centrifuge at 8000 x g for 30 seconds to spin down aggregates.
- Load the supernatant onto an SDS-PAGE gel and electrophorese.
- Transfer to PVDF and probe with appropriate antibodies (Refer to Western blotting Protocol 3 or 4).
NOTE: Phosphotyrosine containing proteins can be selectively purified by loading a Streptavidin:agarose column and eluting the proteins with 10 mM phenyl phosphate.