Human VCP plasmid (TCB197) was subjected to site-directed mutagenesis with primers containing mutations to create each of the indicated variants. Proteins were purified as described.27 (link) Purified VCP (12.5 μL of 50 μM; final concentration in the reaction was 25 nM) was diluted in 20 mL of assay buffer [5 mL of 5× assay buffer A (1× = 50 mM Tris pH 7.4, 20 mM MgCl2, 1 mM EDTA) mixed with 15 mL of water and 25 μL 0.5M TCEP, 25 μL 10% Triton] to make the enzyme solution. 40 μL of the enzyme solution was dispensed into each well of a 96-well plate. The ATPase assay was prepared by adding 10 μL of 1,000 μM ATP (Roche, pH 7.5) to each well and by incubating the reaction at room temperature for 25 minutes. Reactions were stopped by adding 50 μL of BIOMOL Green reagent (Enzo Life Sciences). Absorbance at 635 nm was measured after 4 minutes on the Synergy Neo Microplate Reader (BioTek). All assays were performed in triplicate, and the activity was averaged from independent experiments.