Malachite green ATPase assay

Malachite green (MG) reaction solution was prepared freshly by mixing stock solutions of MG dye [0.111% (w/v) malachite green, 6 N sulphuric acid], 7.5% (w/v) ammonium molybdate and 11% (v/v) Tween 20 in a 10:2.5:0.2 ratio. Samples of purified BiP proteins (5 µM) were incubated in HKM with 3 mM ATP in a final volume of 20 µl for 60 min at 30°C. 15 µl of each sample were then diluted with 135 µl water on a 96-well plate, mixed with 50 µl MG reaction solution and incubated for 2 min at room temperature. 20 µl of a 34% (w/v) sodium citrate solution were added to quench the reactions and after incubation for further 30 min the absorbance was measured at 612 nm with a plate reader (Infinite F500; Tecan). Standard curves were prepared with serial dilutions of KH₂PO₄ and served as a reference to calculate V_max values. Statistical analysis by unpaired t-test was performed using Graphpad Prism version 6.