Protein Kinase Assay with an Immunocomplex

Preparation of Cell Lysate

1. Rapidly rinse control or mitogen-treated cells grown to confluence on 10cm diameter culture dishes with phosphate-buffered saline (PBS) pH 7.4.
2. Lyse the cells with 1.0ml cold lysis buffer (10mM Tris pH 7.4, 1.0% Triton X-100, 0.5% Nonidet P-40, 150mM NaCl, 20mM sodium fluoride, 0.2mM sodium ortho-vanadate, 1.0mM EDTA, 1.0mM EGTA, 0.2mM PMSF) in the culture dish for 30 minutes at 4C with constant agitation.
3. Scrape the cells from the dish and pass the lysate several times through a 26 gauge needle to disperse any large aggregates.
4. Centrifuge for 30 minutes (16,000 x g, 4C). The supernatant is the “total cell lysate”.

Immunoprecipitation of the Protein Kinase

1. Incubate the cell lysate (0.2–1.0mg protein) with 2–5µg soluble antibody.
2. Immunoprecipitate for 1 hour at 4C with end-over-end rotation.
3. If a soluble monoclonal antibody is used, add an equal amount of rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories) and incubate the complexes for an additional 30 minutes at 4C.
4. Wash the desired amount of Protein A:agarose in 1X immunoprecipitation (IP) buffer (1% Triton X-100, 150mM NaCl, 10mM Tris pH 7.4, 1mM EDTA, 1mM EGTA pH 8.0, 0.2mM sodium ortho-vanadate, 0.2mM PMSF, 0.5% NP-40), followed by a 4 minute centrifugation (16,000 x g, 4ŢC). Bring the Protein A:agarose to a 50% suspension in 1X IP buffer.
5. Incubate the immune complexes from step 2 or 3 for 30 minutes at 4C with 10µl of the 50% Protein A suspension.
6. Wash the complexes by resuspension in IP buffer, followed by a 4 minute centrifugation (16,000 x g, 4C). Repeat the wash at least twice.
7. Collect the complexes by centrifugation for 3 minutes (16,000 x g, 4C).

Kinase Assay

1. Wash the immunocomplexes three times at 4C with kinase buffer (10mM Tris pH 7.4, 150mM NaCl, 10mM MgCl₂, 0.5mM DTT).
2. Remove the supernatant by aspiration. Incubate the pellets for 15 minutes at 37C with 40µl of the kinase buffer that contains 25µM ATP, 2.5 µCi [³²Pg]ATP, and the appropriate protein substrate* at 1.0 mg/ml.
3. Add 15µl of boiling 5X concentrated electrophoresis sample buffer (312.5mM Tris pH 6.8, 10% SDS, 25% glycerol, 0.015% bromophenol blue, 5% b-mercaptoethanol) to terminate the reaction. Boil for an additional 5 minutes.
4. Centrifuge the samples and electrophorese the soluble fractions.
5. Fix and stain the gel (0.25% Coomassie blue in 45% methanol, 10% acetic acid), destain (40% methanol, 10% acetic acid), dry, and expose to X-ray film. Kinase activity will be indicated by a band of phosphorylated protein substrate.