Protein Kinase Assay after Denaturation and Renaturation
Preparation of the Cell Lysate
- Rinse a 60mm culture dish of confluent cells with phosphate-buffered saline (PBS).
- Add 0.5ml of boiling lysis buffer (1% SDS, 1.0mM sodium ortho-vanadate, 10mM Tris pH 7.4).
- 3. Transfer lysate to a 1.5ml microcentrifuge tube and boil the samples for an additional 5 minutes. Sonicate lysate briefly or pass several times through a 26 gauge needle to shear nucleic acids. This is the “total cell lysate”.
Immunoprecipitation and Electrophoresis
- Immunoprecipitate MAP kinase (ERK) as indicated in Protocol 7 (denaturing conditions).
- Prepare a polyacrylamide gel containing 1.0 mg/ml of myelin basic protein or the EGF receptor peptide (amino acids 663–683) in the resolving gel. The stacking gel is prepared without the substrate. Electrophorese the immunoprecipitated ERKs alongside appropriate molecular weight standards.
Removal of SDS
- After electrophoresis, wash the gel with 20% 2-propanol in 50mM Tris pH 8.0 for 1 hour at room temperature with continuous agitation, changing the wash buffer at least twice (This removes SDS from the gel).
- Incubate the gel for 1 hour in 250ml of buffer A (50mM Tris pH 8.0, 5mM DTT) with continuous agitation.
Incubate the gel for 1 hour at room temperature in a solution of 6M guanidine-HCl in buffer A with continuous agitation.
Incubate the gel in buffer A containing 0.04% Tween-40 for 16 hours at 4C with continuous agitation and at least three buffer changes.
- Pre-incubate the gel in kinase assay buffer (40mM Hepes-HCl pH 8.0, 2.0mM DTT, 0.1mM EGTA, 5mM magnesium acetate) at room temperature with continuous agitation.
- For the kinase assay, incubate the gel for 1 hour at room temperature in kinase assay buffer containing 5–50 µCi [32Pg]ATP. Make certain the entire gel is covered with the reaction mixture, which usually requires 15–20ml for a 14cm gel. This volume can be reduced if the reaction is done in a plastic bag.
- Wash the gel at least three times with 5% (w/v) trichloroacetic acid and 1% sodium pyrophosphate. The washes should be performed repeatedly for about 1.5 hours to reduce the background.
- Dry the gel under vacuum on 3MM paper and expose to X-ray sensitive film and intensifying screen at -80C. ERK activity will be indicated by the phosphorylation of myelin basic protein adjacent to the ERK (Molecular weight 42–44kDa).
Campos-González, R. and Glenney, Jr., J.R. 1992 J. Biol. Chem. 267:14535
Kameshita, I. and Fujisawa, H. 1989 Anal. Biochem. 176:22