Table 1.

Different methods used to measure HSP70 ATPase activity

Assay	Advantages	Limitations	Method of detection
Radiometric29–32	• Sensitive	• Uses radioactive materials • Lack of specificity • HTS: No	• Radiolabeled [32Pi] release due to ATPase activity measured by thin layer chromatography
Colorimetric phosphate detection18,33–35	• More sensitive than radiometric assay • No use of radioactive materials • Few steps, cost-effective, good automation • HTS: Yes	• Low signal • Not usable with 384/1536 well plates • Lack of specificity	• Phosphate released due to ATPase activity binds to molybdenum ions, and then color is developed by reducing agents such as stannous chloride, ascorbic acid, and dyes (malachite green, crystal violet, and quinidine green).
Fluorescence35,36	• More sensitive than absorption-based assays • Proteins with low intrinsic activity can be used • Cost-effective and sensitive • HTS: Yes	• Autofluorescence of molecules can lead to false-positive molecules • Lack of specificity	• Reads fluorescence from plastic plates that is dependent on the amount of colored complex between phosphate released due to ATPase activity.
Fluorescence polarization37,38	• Potential assay to distinguish ATP modulators • HTS: Yes • Not used in HTS yet	• Depends on substrate	• F ITC-labeled substrate binds to the ADP-bound HSP70 form as a result of ATPase activity and fluorescence is changed.
Enzyme-linked immunosorbent assay35,45			• Denatured substrate is coated on plate. It traps ADP-bound HSP70. HSP70 detected by antibodies.
Tryptophan fluorescence35,46		• Low signal difference • Lack of specificity	• Conformational changes caused by hydrolysis can be detected by measuring tryptophan fluorescence.
Luciferase refolding39–44	• Sensitive • HTS: Yes	• Indirect method • Lack of specificity • HSP70 ATPase activity inhibitors would appear as false negatives because they do not show an effect on refolding.	• Refolded luciferase is a result of HSP70 ATPase activity that reacts with luciferin and emits light (bioluminescence).

Notes: Currently available methods for detection of HSP70 ATPase activities are tabulated. Lack of specificity refers to a lack of specific identification of molecules that affect AD, ie, beta amyloid and tau proteins.

Abbreviations: AD, Alzheimer’s disease; ADP, adenosine diphosphate; ATP, adenosine triphosphate; FITC, fluorescein isothiocyanate; HSP70, heat shock protein 70; HTS, high throughput screening; Pi, phosphate.