Benchmark fluorescent protein standard

The membrane suspensions of the wild type and each mutant were adjusted to a protein concentration of 5 mg/mL. Fifteen microliters of the membrane suspension (5 mg/mL) was mixed with an equal volume of 2 × sample buffer (50 mM Tris–HCl pH 7.5, 5 mM EDTA, 5% β-mercaptoethanol, 5% glycerol, 4% SDS, 0.02% bromophenol blue, 2 × concentration of complete EDTA-free protease inhibitor cocktail), and 20 μL of the mixture was applied to the SDS-PAGE gel of the Tris–glycine buffer system without boiling the sample. One microliter of the Benchmark Fluorescent Protein standard (Thermo Fisher Scientific, USA) was applied to the gel as a molecular size marker. Electrophoresis was performed at 100 V and 4 °C. Fluorescence images were obtained using Typhoon FLA 7000 (GE Healthcare, USA).

The protein solutions were mixed with SDS-PAGE sample buffer (final concentration of components: 2% SDS, 10% (w/v) glycerol, 0.002% bromophenol blue, 50 mM Tris-HCl, pH 6.8) to prepare the samples for SDS-PAGE analysis. To denature the proteins, the mixed solution was heated at 95 °C for 5 min. Samples were subjected to SDS-PAGE using SuperSep Ace precast gels (Wako Pure Chemical Industries, Japan) and the running buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS). 15% gels were used unless otherwise indicated. Visualization of fluorescent bands and the image capturing were carried out on a ChemiDoc XRS + Imaging System (BIO-RAD) equipped with a bandpass filter for GFP detection (520 nm, full-width half-maximum [FWHM] = 20 nm, Bio-Rad) under UV illumination. For in-gel fluorescent detection, BenchMark™ Fluorescent Protein Standard (ThermoFisher, product # LC5928) was used as marker proteins. After detection of the fluorescent bands, the gels were stained with CBB Stain One (Nacalai Tesque). Gel image was captured using a ChemiDoc XRS + Imaging System.