Sypro orange stock

For the 96-well refolding detection assay, a 25x dilution of the 5000x SYPRO Orange stock (Invitrogen) was prepared in water. For each well, 5 μL of this diluted SYPRO Orange and 45 μL of the refolding sample was added. The plate was covered with sealing tape and centrifuged at 600 g for 1 min to remove any air bubbles. A BioRad CFX96 RT-PCR machine was programmed with a 3 min equilibration time at 22 °C. Subsequently, the plate was heated from 22 to 90 °C with stepwise increments of 0.5 °C per 30 s, followed by the fluorescence reading optimized for SYPRO Orange at 485 nm (Excitation) and 530 nm (Emission) after each step. The 200 μL refolding sample prepared allows for the refolding experiments to be analyzed by DGR at four different time points, aiding the identification of the optimal incubation time for the refolding process.

DSF^{25 (link)} experiments were performed using a Roche LightCycler 480 II in sealed Axygen 384-well plates (PCR-384-LC480). Reactions (20 μL) contained 0.1 mg/mL TKBD-LHR-RING and full-length Cbl-b, in 30 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM TCEP, and SYPRO Orange (5000× SYPRO Orange stock in DMSO, Invitrogen). C7683 was tested at a serially diluted range from 0.01 to 100 µM with a final concentration of 2% DMSO. Thermal denaturation was monitored from 20 to 95 °C at a rate of 4 °C increase per minute, and data points were collected at 0.5 °C intervals.