Luciferase Refolding Activity Assay

Protein refolding activity was examined with luciferase inactivation assay with minor modifications [72 (link)]. In brief, 100 nM luciferase (Sigma-Aldrich Cat#61970-00-1) was heated at 42 °C alone or in the presence of the fly muscle lysates in luciferase refolding buffer (LRB: 25 mM HEPES-KOH at pH 7.4, 150 mM potassium acetate,10 mM magnesium acetate and 10 mM DTT). Luminescence was measured on a microplate reader (Synergy HTX, BioTek, Winooski, Vermont, USA) at different time points (5, 10, 15 min). Luminescence values were then normalized with protein concentrations using BSA as a standard by bicinchoninic acid (BCA) protein determination kit (YEASEN Cat#20201ES76) according to the manufacturer’s instructions.