Coomassie brilliant blue g 250

The integration of the ion channels into the liposomes was evaluated by using iohexol density gradient centrifugation. Three hundred µL of the purified proteoliposome suspension prepared in Section 2.1 was mixed with the same volume of 80% iohexol (TCI, Tokyo, Japan)/PBS solution to obtain a final concentration of 40% iohexol. As shown in Figure 1A, 600 μL of 40% iohexol/proteoliposome/PBS, 650 μL of 35% iohexol/PBS, 650 μL of 30% iohexol/PBS, and 100 μL of PBS were layered in a 2 mL ultracentrifuge tube. Ultracentrifugation was performed at 206,000× g and 20 °C for 1 h. Subsequently, two hundred µL of the solution were collected from the top and bottom of the tube, respectively. The samples taken from the crude translation reaction mixture, centrifugation-purified proteoliposomes, and the top and bottom fractions of iohexol density gradient centrifugation were subjected to SDS-PAGE analysis. SDS-PAGE gels were stained using Coomassie Brilliant Blue G-250 (Nakarai tesque, Kyoto, Japan).

Gel-shift assays were performed by optimizing the method reported in previous studies [44 (link),49 (link)]. Reduced and denatured RNase A (16 µM) was incubated with refolding buffer containing 50 mM HEPES (pH 7.2), 150 mM NaCl, 1 mM reduced glutathione (GSH), and 0.2 mM oxidized glutathione (GSSG), in the absence/presence of 1 µM PDIs or 0.5 µM P5 and other PDIs. At selected time points, the reaction was quenched, and free thiols were modified by addition of Laemmli 4 × sodium dodecyl sulfate (SDS)-sample buffer [32 (link)] containing 10 mM methoxy-polyethylene glycol maleimide MW 2000 (mPEG-mal 2k; Cat. No. ME-020MA; NOF America Corporation, White Plains, NY, USA). Modified samples were separated by nonreducing 14% SDS-polyacrylamide gel electrophoresis (PAGE) using a WIDE RANGE gel (Nacalai Tesque). Proteins were stained with Coomassie Brilliant Blue G250 (Nacalai Tesque) [50 (link)]. Band intensities were analyzed using ImageJ/Fiji [46 (link)] and Microsoft Excel. The relative band intensities of fully reduced form (R) and fully oxidized form (N/4SS) on SDS-PAGE gels were calculated compared with the band intensity of negative controls (0) and positive controls (N) in the same gels, respectively.