Assessing CaMKIIα Autophosphorylation

To assess the ability of CaMKIIα WT and mutants to autophosphorylate at T286, the kinases were added at a final concentration of 20 nM to a reaction buffer containing 50 mM PIPES pH 7.2, 0.1% BSA, 2 mM CaCl₂, 10 mM MgCl₂, 100 mM ATP, 1 μM microcystin-LR, 100 nM calmodulin. Kinases were reacted at 30 °C for 30 seconds, or as indicated. Reactions were stopped by addition of gel loading buffer (2% SDS, 50 mM dithiothreitol, 67.5 mM Tris pH 6.8, 10% glycerol, 0.16 mg ml⁻¹ bromophenol blue) and boiling for 5 min. Samples were loaded on 10% SDS–polyacrylamide gel electrophoresis gels then transferred to polyvinylidene difluoride membranes42 (link)43 . Blots were blocked in 5% milk then probed with anti-phospho-T286 CaMKII antibody (Phosphosoutions) diluted 1:3,000 in 1% milk. Images were acquired on an Alpha Imager (Alpha Innotech) after exposure to Western Lightning ECL reagent (Perkin Elmer) and quantified42 (link)43 44 (link).