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Assay Method Information

Assay Name:  CaMKII delta Biochemical Assay
Description:  The biochemical activity of recombinant CaMKIIδ was measured by monitoring the phosphorylation of a target peptide using Homogenous Time Resolved Fluorescence (HTRF) technology from CisBio Bioassays. 10 pM CaMKIS (Life Technologie Corporation) was incubated for 1 hour with 110 nM calmodulin (EMD Millipore), 1 μM Biotin-STK1 (target peptide, CisBio Bioassays) and 12 μM ATP (Sigma-Aldrich) in a buffer containing 20 mM Hepes pH7.2, 10 mM MgCl2 and 1 mM CaCl2 as well as the compound to be tested or DMSO at a final concentration of 0.6%. The reaction was stopped with the addition of the detection buffer (containing EDTA) with Eu-cryptate labeled anti-phospho-STK1 antibody (CisBio Bioassays) and 125 nM XL665 labeled streptavidin (CisBio Bioassays). After a 1 hour incubation, fluorescence at 620 nm and 665 nm was read using an EnVision Multilabel Reader (Perkin Elmer) and the ratio of signal at 665 nm and 620 nm (665/620) was calculated and plotted against compound concentration to determine compound IC50.
Affinity data for this assay
 

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Last update November 1, 2007
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