Heat Shock Protein 70 (HSP70) is a vital and highly conserved protein. It exists in organisms from bacteria to humans. Under normal conditions, it aids in protein folding and maintenance of cellular proteostasis. When cells encounter stress like heat or toxins, HSP70 expression surges. It then prevents protein aggregation, assists in refolding damaged proteins, and plays a key role in cell survival and stress response, safeguarding cellular function and integrity.Besides,The activation of HSP70 protein depends on the hydrolysis of ATP, which can produce inorganic Pi phosphate. Thus,the activity of recombinant human HSP70 was measured by its ability to hydrolyze the substrate ATP to phosphate which was detected by the malachite green phosphate detection Kit (Beyotime # S0196M). The reaction was performed in the assay buffer 20 mM HEPES, 13 mM NaCl and 1% glycerol, pH 8.5, initiated by addition 27 μL of various concentrations of HSP70 (diluted by assay buffer) to 3 μL of 100 mM ATP and 100 mM MgCl2 mixture. The final well serves as a negative control with no HSP70, replaced with 27 μL assay buffer. Incubated at 37℃ for 5min, then stop the reaction by quickly freezing samples in the dry ice and ethanol. 4 ul samples was added to 196 ul assay buffer after thawing at room temperature followed by adding 70 ul phosphate detection agent and incubated at room temperature for 30min, then read at a wavelength of 630 nm. The specific activity of recombinant human HSP70 is >12000 pmol/min/μg.