Native polyacrylamide gel electrophoresis (native-PAGE)

Native-PAGE was performed as described (Preissler et al., 2015). Tris-glycine polyacrylamide gels consisting of a 4.5% stacking gel and a 7.5% separation gel were used to separate purified protein or proteins from mammalian cell lysates under non-denaturing conditions to detect BiP oligomers. The gels were run in a Mini-PROTEAN electrophoresis chamber (Bio-Rad) in running buffer (25 mM Tris, 192 mM glycine, pH ~8.8) at 120 V for 2 hr when cell lysates were applied or for 1:45 hr when His6-tagged purified BiP proteins were analyzed. The proteins were then visualized by staining with InstantBlue Coomassie solution (expedeon, UK) or transferred for immunodetection to a polyvinylidene difluoride (PVDF) membrane in blotting buffer (48 mM Tris, 39 mM glycine, pH ~9.2) containing 0.04 (w/v) SDS for 16 hr at 30 V. The membrane was washed after the transfer for 20 min in blotting buffer supplemented with 20% (v/v) methanol before blocking. Seven µg of purified BiP protein was loaded per lane on a native gel to detect BiP oligomers by Coomassie staining and volumes of lysates corresponding to 30 µg of protein (CHO-K1 and AR42j cells) or 90 µg protein (Flp-In T-REx 293 cells) were loaded per lane.