Instantblue coomassie solution

A 50 μL mixture of 10 μM Noc (WT/mutants) ± 1 mM NTP ± 1 μM 22-bp NBS/parS dsDNA was assembled in a reaction buffer [10 mM Tris-HCl pH 7.4, 200 mM NaCl, and 1 mM MgCl₂] and was incubated for 10 min at 22°C or for 1, 5, 10, 15, and 30 min at 4°C. Subsequently, BMOE was added to the final concentration of 1 mM, and the reaction was quickly mixed by three pulses of vortexing. SDS-PAGE sample buffer containing 23 mM β-mercaptoethanol was then added immediately to quench the crosslinking reaction. Samples were heated to 50°C for 10 min before being loaded on 12% WedgeWell Tris-Glycine polyacrylamide gels (Thermo Fisher). Each experiment was triplicated. Polyacrylamide gels were stained in an InstantBlue Coomassie solution (Abcam) and band intensity was quantified using Image Studio Lite (LI-COR Biosciences). The crosslinked fractions were averaged, and their standard errors were calculated in Excel.
CTPɣS was custom synthesized either in-house (Rejzek and Le, 2021 (link)) or by Jena Biosciences.

Native-PAGE was performed as described (Preissler et al., 2015 (link)). 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.