White 96 well rt pcr plate

Thermal shift assays were performed as described by Ericsson et al.24 20 μL of ~13 μM Irc15p protein solution was pipetted into a white 96‐well RT‐PCR plate from Bio‐Rad (California, USA) both at different pH, in the absence and presence of 150 mM NaCl and in buffer B in the presence and absence of a final concentration of 50 mM NADH, 50 mM NAD⁺, 50 mM NADPH, 50 mM NADP⁺, or 50 mM sodium dithionite. Two μL of a 1:500 dilution of SYPRO® orange from Molecular Probes (Oregon, USA) was added. The plates were sealed with an Optical‐Quality Sealing Tape from Bio‐Rad (California, USA) and heated in a CFX Connect™ Real‐Time PCR detection system from Bio‐Rad (California, USA) from 20 to 95 °C in increments of 0.5 °C/5 s. Fluorescence changes of the dye were detected at a wavelength between 470 and 500 nm. Melting temperatures (T_m) were determined using CFX Manager 3.0 software from Bio‐Rad (California, USA).

The melting point determinations were performed as described by Forneris et al. and Ericsson et al. 22, 23. Briefly, 150 μm NQO1 WT or P187S variant in 50 mm HEPES at pH 7.0 with or without BPPSA at a concentration of 2 mm were mixed in a white 96‐well RT‐PCR plate (Bio‐Rad Laboratories, Hercules, CA, USA) sealed with optical‐quality sealing tape (Bio‐Rad Laboratories). The plate was heated from 20 to 95 °C, with 0.5 °C steps, in a CFX Connect™ Real‐Time PCR Detection System from Bio‐Rad Laboratories, and the change in fluorescence due to the release of the FAD cofactor upon unfolding of the protein was detected. The thermal stability was analyzed using the cfx manager 3.0 software from Bio‐Rad Laboratories.