ATPase assays with malachite green

Active compounds from the primary screen were confirmed by subsequent ATPase assays using MG, as described previously (36). Briefly, in a clear 96-well plate, compounds were incubated with Hsp72 and co-chaperones at specified concentrations in 25-μl total volume with a final DMSO concentration of 4%. All malachite green assays were performed in an assay buffer composed of 100 mm Tris, 20 mm KCl, 6 mm MgCl₂, 0.01% Triton X-100, pH 7.4. The reaction was initiated by the addition of ATP at a final concentration of 1 mm and incubated at 37 °C for 1 h. After incubation, 80 μl of MG reagent was added, followed by 10 μl of saturated sodium citrate to quench the reaction, and absorbance of 620 nm was measured on a SpectraMax M5 plate reader (Molecular Devices). ATP hydrolysis rates were calculated by comparison to a phosphate standard.