Assay Report Card

ID: ALA3888750

Type: Binding

Description: 33P-Based Assay: The biochemical EC50 (half-maximal concentration required for full activation) of compounds for the activation of AMPK was evaluated by 33P-based assay using SAMS peptide (commercially available) derived from ACC-1. Twenty μl of phosphorylated AMPK diluted in assay buffer, (50 mM HEPES, 1 mM EGTA, 10 mM MgCl2, 0.25 mM DTT, 0.01% Tween-20, 0.01% BSA (pH 7.5)) was added to 384 well plates containing 1 μL of test compound. Following a fifteen minute room temperature incubation, 10 μL of protein phosphatase PP2A was added to the plate to dephosphorylate pThr172 of AMPK. After incubation for 90 minutes, 10 μL of substrate mixture containing 41 nM okadaic acid, 82 μM SAMS peptide, 82 μM ATP and tracer amounts (6.8 nM) of 33P-containing ATP was added to the plate. The reaction was terminated after 60 minutes incubation at room temperature by the addition of 15 μL of 2% H3PO4. Subsequently, 45 μl of reaction mix was transferred to 384 well Millipore MZPH filter plates (MZPHNO50) pre-treated with 25 μL of 2% H3PO4 and the plates were washed three times with assay buffer. 20 μL of Ready Safe scintillation fluid was added to dried plates followed by detection on the Trilux detector.

Organism: Homo sapiens