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Assay Method Information |
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| | Inhibition Assay |
| Description: | Compounds were added in 5 uL volume to wells in UV transparent 96-well plates ( area well size). The final compound concentrations tested ranged from 0.5 nM to 10 uM). Assays were performed in duplicate. CaMKIIδ is added to at a final concentration of 16 nM to a mixture containing 100 mM Tris (pH 7.5), 150 mM KCl, 0.27 mM EGTA, 1.3 mM PEP, 0.2 mg/ml AC3, 6.9% (v/v) PK/LDH mixture (Sigma P0294), 0.38 mM NADH and kept on ice. 72 uL of the enzyme mixture was added to the wells containing compounds and the plate was shaken briefly and kept on ice. The assay was initiated by adding 23 uL of a mixture containing 100 mM Tris (pH 7.5), 150 mM KCl, 1.7 mM CaCl2, 48 mM MgCl2, 0.35 mM ATP and 6.7 ug/mL calmodulin. The rate of ADP released was measured as the rate of absorbance decrease at 340 nM at 25° C. |
| Affinity data for this assay | |
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