Table 4.

Summary of Hsp70 Adenosine Triphosphatase Activity Assay

Step	Parameter	Value	Description
1	Compound addition to plate	0.6 μL	0.5 or 2.5 mM compound in DMSO added to 384-well plates
2	Protein addition	24.4 μL	1600 nM Hsp70 in 50 mM HEPES, pH 7.2, containing 2 mM MgCl2
3	Incubation time	15 min	On ice
4	ATP addition	5 μL	6 mM ATP in assay buffer
5	Incubation time	1 h	Lidded, 32°C
6	Malachite reagent addition	7.5 μL	PiColorLock™ Gold mixture
7	Incubation time	5 min	Lidded, ambient temperature
8	Stabilizer addition	3 μL
9	Assay Readout	630 nm	Absorbance

1. 0.5 or 2.5 mM compound used for 10 and 50 μM final concentrations, respectively.

8. Do not directly mix P_iColorLock™ Gold with stabilizer.

 ATP, adenosine triphosphate.