Recombinant Rat CaMKII was expressed in Insect cells.
Calmodulin-Dependent Protein Kinase II (CaMKII) is a serine/threonine kinase. It is a Ca2+/calmodulin-dependent, truncated monomer (1-325 amino acid residues) of the α subunit. Autophosphorylation of threonine 286 in the presence of Ca2+ and calmodulin activates CaMKII and produces substantial Ca2+/calmodulin-independent activity.
CaMKII has been purified to >95% homogeneity as determined by SDS-PAGE and Coomassie Blue staining.
One unit is defined as the amount of activated CaMKII required to catalyze the transfer of 1 pmol of phosphate from ATP(200mM) to Autocamtide-2 (CaMKII Peptide Substrate), KKALRRQETVDAL (50 µM), in 1 minute at 30 centigrade in a total reaction volume of 30 µl.
Optimal incubation times and enzyme concentrations must be determined empirically for each particular substrate.If the objective is to measure enzyme activity using gamma-labeled ATP, it is best to use 100-200 μM ATP in order to have higher specific activity of gamma-labeled ATP (100-500 cpm/pmol). Also, an excess of substrate should be used, and the level of phosphorylation should not exceed 10% for determination of the initial rate. To phosphorylate a protein or peptide substrate to completion, the ATP concentration should be about 5-fold over the limited substrate concentration. Higher enzyme concentration and prolonged incubation times should be employed (4). Apparent Km values of ATP for most protein kinases are below 100 μM.If the source of protein to be phosphorylated is a crude extract of cells or tissue, it is very important to include the appropriate protease and protein phosphatase inhibitors in the lysis buffer and to use shorter incubation time for phosphorylation.
Heat Inactivation: 65 centigrade for 20 min
Store product at –70 centigrade. Avoid repeated freeze/thaw cycles.