Novex sharp unstained protein standard

Samples were obtained from cells grown on LB plates at 37°C for 10 h. The populations were harvested by centrifugation at 21,600×g for 1 min at 4°C. The harvested cells were washed three times with 0.1 M Tris–HCl (pH 7.0) buffer. The washed cells were re-suspended and sonicated for 20 min (20 s treatment was repeated with 20-s intervals) at 4°C with a Bioruptor UCD-250 (Cosmo Bio Co. Ltd.; Tokyo, Japan). The disrupted cells were centrifuged at 21,600×g for 10 min at 4°C to remove cell debris and then subjected to ultracentrifugation at 166,000×g for 60 min at 4°C to remove the insoluble fraction. The soluble fraction was used in SDS-PAGE analysis using the NuPAGE SDS-PAGE Gel system (Invitrogen). Proteins were separated by electrophoresis using 4–12% Bis–Tris NuPAGE gels with MES running buffer, and then stained with Coomassie Brilliant Blue. Protein concentration in the soluble fraction was determined using the Pierce 660 nm Protein Assay Kit (Thermo Fisher Scientific Inc.; Waltham, MA, USA). The molecular weight marker was Novex^™ Sharp Unstained Protein Standard (Invitrogen).