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10% Ammonium persulfate (APS) (#161-0700; Bio-Rad). 9. 3. 10. 005% bromophenol blue. 11. Destaining solution: 15% isopropanol, 7% acetic acid. 3. Western Blot Analysis 1. 2. 3. 4. 5. 6. 7. 8. 9. Transfer apparatus. 8. Nitrocellulose membrane (Protran BA 85; Schleicher & Schuell). Whatman paper (3 mm). 05% Tween-20. Blocking solution: 2% (w/v) BSA in washing buffer. , monoclonal antidiphospho-ERK [M-8159] and polyclonal anti–general ERK [M-5670] from Sigma, Israel), and secondary antibody (alkaline phosphatase [AP]–or horseradish peroxidase [HRP]–conjugated antimouse or antirabbit Fab antibodies from Jackson) diluted in washing buffer to appropriate dilutions.
End the phosphorylation reactions by adding 10 µL of 4X sample buffer to each tube. Boil, centrifuge (1 min at 14,000g), and load the supernatants on a 15% SDS-PAGE gel. 5 cm from the bottom of the gel, stop the current. To remove the excess free radiolabeled ATP, which migrates just in front of the bromophenol blue, cut out the part of the gel below the dye. This will considerably reduce the amount of radioactivity in the gel. 3.. Wash briefly with distilled water and let dry. An alternative way would be to stain, destain, and dry the gel on a Whatman 3-mm paper, but this procedure does not allow further detection of proteins in the gel as described for the immunoprecipitated ERKs in step 16.