Firefly Luciferase Disaggregation Assay

Firefly luciferase aggregates were formed by incubating luciferase (50 µM) in LRB (pH=7.4) plus 8M urea at 30°C for 30 min. The luciferase was then rapidly diluted 100-fold into LRB, snap frozen, and stored at −80°C until use. Hsp104 and Skd3 variants (1 µM monomer, unless otherwise indicated) were incubated with 50 nM aggregated firefly luciferase in the presence or absence of Hsc70 and Hdj2 (0.167 µM each) in LRB plus 5 mM ATP plus an ATP regeneration system (ARS; 1 mM creatine phosphate and 0.25 µM creatine kinase) at 37°C for 90 min (unless otherwise indicated). The nucleotide-dependence of Skd3 disaggregation activity was tested in the presence of ATP (Sigma), AMP-PNP (Roche), ATPγS (Roche), or ADP (MP Biomedicals) for 30 min at 37°C without ARS. Recovered luminescence was monitored in Nunc 96 Well Optical plates using a Tecan Infinite M1000 plate reader (DeSantis et al., 2012 (link)).