2.4. ATPase activity of Hsp70

An ATPase assay was adapted from Ko et al.²⁶ Briefly, a calibration curve of phosphate (0.625 μM – 50 μM) with assay buffer (100 mM Tris-HCl, 20 mM KCl, 6 mM MgCl₂, pH 7.4) was made with and without ATP. For samples, Hsp70 (2 μM) was added to Tris buffer. The reaction was initiated by addition of ATP (4 mM) and shaken at 37 °C for 15 minutes, followed by incubation with BiolMol Green (Enzo Life Sciences) at rt for 20 minutes. Absorbance of each well was measured at 625 nm. Calibration samples with and without ATP were each run in duplicate and samples with Hsp70 were run in triplicate.