Assay Method Information

Assay Name:  ACCase-Coupled Enzyme Assay
Description:  Assays were performed in 384-well clear bottom plates (Corning; catalog no. 3702), that contained inhibitor solvated in DMSO. To each well of the plate consisting of 50 mM Hepes (pH 8.0), 100 mM KCl, 1 mM TCEP, 5 mM MgCl2, 0.1 mg/mL BSA, 0.005% (vol/vol) Tween 20, 30 nM E. coli BC, 50 nM E. coli CT, 50 nM biotinylated E. coli BCCP, and 0.5 unit/mL E. coli purine nucleoside phosphorylase was added. After 5 min of coincubation of inhibitors with solution 1, the ACCase reaction was initiated by addition of 40 uL of solution 2, which consisted of 500 uM citrate (pH 4.2), 150 uM 7-methyl-6-thioguanosine (MESG; Berry and Associates), and 0.005% (vol/vol) Tween 20. Reaction progress was monitored by the increase in absorbance at 360 nm. Inhibitor potency was assessed by duplicate 20-point titrations of inhibitor from 96 uM to 9.6 nM. Inhibition data were fit to the standard IC50 equation.
Affinity data for this assay
 

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