Hsp70 hsp40 glow fold protein refolding kit

CD spectra of WT and T246M U-box CHIP were collected as previously described [54 (link)] at 0.25 mg/mL and 15 °C in 10 mM sodium phosphate (pH 7.0) with 20 mM NaCl and 1 mM DTT. The T_m for WT and T246M U-box CHIP was determined by monitoring the temperature dependence of CD at 222 nm.
Luciferase refolding: The HSP70/HSP40 Glow-Fold Protein Refolding Kit (Boston Biochem) was used per the manufactures instructions. The luciferase was heat denatured for 15 min at 42 °C. CHIP proteins or IgG were added to the reactions at a final concentration of 0.28 mg/ml in the absence or presence of HSP40/70 and incubated for 60 min at 30 °C. Activity was measured by luminescence and data normalized to heat denatured luciferase in the absence of any additional proteins.

The Glow-Substrate peptide was unfolded by heating at 40 °C for 7 min and subjected to a refolding assay using the HSP70/HSP40 Glow-Fold Protein Refolding kit (BostonBiochem) according to the manufacturer’s instructions. For the quantification of the refolded amounts of peptide, the luciferase signal was measured by a luciferase reader (GloMax, Promega) at various reaction times (10, 20, 30, 40, 50, and 60 min). The experiments were independently repeated three times.

To investigate the effect of GO‐Y030 on HSP70/HSP40‐mediated chaperone activity in the cell‐free system, we employed an HSP70/HSP40 Glow‐Fold Protein Refolding Kit (Boston Biochem, Bio‐Techne Corp.) and performed an assay in accordance with the manufacturer's protocol with modifications. In brief, Glow‐Fold Substrate protein equivalent to luciferase protein was mixed with HSP70/HSP40 complex, ATP, and the compounds to be examined or dimethylsulfoxide alone. The concentration of the HSP70 inhibitors was as: PFT‐μ, 0.8 mm; VER‐155008, 0.4 mm; and JG98, 5 μm. The reactions were exposed to 45 °C for 7 min and then placed in 30 °C to initiate refolding reactions. After the indicated times, aliquots taken from each of the reactions were mixed with luciferin reagent and luciferase activity was measured using a TECAN Infinite 200 microplate reader. Average luminescence was measured as counts per second from three independent experiments and the data are presented as a percentage of the refolding activity. The refolding activity of each sample before heat shock was set as 100%.