Protein Kinase Assay with an Immunocomplex
Preparation of Cell Lysate
- Rapidly rinse control or mitogen-treated cells grown to confluence on 10cm diameter culture dishes with phosphate-buffered saline (PBS) pH 7.4.
- 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.
- Scrape the cells from the dish and pass the lysate several times through a 26 gauge needle to disperse any large aggregates.
- Centrifuge for 30 minutes (16,000 x g, 4C). The supernatant is the “total cell lysate”.
Immunoprecipitation of the Protein Kinase
- Incubate the cell lysate (0.2–1.0mg protein) with 2–5µg soluble antibody.
- Immunoprecipitate for 1 hour at 4C with end-over-end rotation.
- 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.
- 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.
- Incubate the immune complexes from step 2 or 3 for 30 minutes at 4C with 10µl of the 50% Protein A suspension.
- 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.
- Collect the complexes by centrifugation for 3 minutes (16,000 x g, 4C).
- Wash the immunocomplexes three times at 4C with kinase buffer (10mM Tris pH 7.4, 150mM NaCl, 10mM MgCl2, 0.5mM DTT).
- 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.
- 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.
- Centrifuge the samples and electrophorese the soluble fractions.
- 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.
*Protein Substrates commonly used for specific kinases
Histone H1 (Sigma)
Annexin I, Angiotensin (Sigma)
ERK1 & ERK2
Myelin Basic Protein