J 820 cd spectrometer

CD spectra were recorded on a model J-820 CD spectrometer (JASCO). Far-UV CD measurements were performed with 0.5 mg/mL of IFNγR in PBS using a 1-mm cell and a bandwidth of 1 nm. Spectra were recorded five times for each sample.

For CD analyses, Vivaspin 20 columns (10 kDa molecular weight cutoff, GE Healthcare) were used to exchange purified McpB LBD into CD buffer (20 mM sodium phosphate, 100 mM NaCl, pH 7.0). CD experiments were performed on a Jasco J-820 CD spectrometer (Tokyo, Japan) equipped with a 1-mm path length cuvette using 20 μM McpB LBD in the absence and presence of 100 μM boric acid. CD spectra (190–260 nm) were recorded at 25 °C. For thermal denaturation experiments, CD at 222 nm was monitored from 20 to 70 °C.

α-synuclein samples in the presence or absence of Hsp60 GW (or WT) were diluted to a final concentration of 0.2 mg/mL in 25 mM Tris buffer (pH 7.7 at 25 °C). For α-synuclein samples in the presence of Hsp60 GW (or WT), spectra of Hsp60 GW (or WT) were subtracted to obtain the net spectra of α-synuclein. Stability of Hsp60 AD was analyzed using 0.05–0.1 mg/mL proteins with buffer containing 25 mM Tris buffer (pH 7.7 at 25 °C) and 150 mM NaCl. Hsp60 AD samples were incubated overnight at 4–37 °C. The samples were measured on a J-820 CD spectrometer (JASCO, Tokyo, Japan) using a 0.1 cm path length synthetic quartz cuvette (GL Sciences, Tokyo, Japan). CD spectra (average of 16 spectra) were corrected for buffer signals.