Mini protean tgx 7.5 precast gels

Homemade Tris-HCl 4–20% polyacrylamide gradient gels or Mini-PROTEAN TGX 7.5% precast gels (BioRad) were used with Tris-glycine running buffer. 2× SDS sample buffer refers to a mixture containing 125 mM Tris (pH 6.8), 4% (wt/vol) SDS, 30% (vol/vol) glycerol, 0.005% bromophenol blue, and 5% (vol/vol) β-mercaptoethanol. SDS-PAGE analyses were performed at 200 V for 45 to 60 min. To analyze purified protein complexes for cryo-EM, SDS-PAGE was performed and followed by staining with Coomassie Brilliant Blue (Alfa Aesar). Coomassie-stained SDS-PAGE gels were imaged using the “Gel” feature of an Azure Biosystems C400 imager. For Western blotting, proteins were transferred onto Immun-Blot PVDF membranes (Bio-Rad) and then incubated with appropriate antibodies. All HRP conjugates were visualized with the Amersham ECL Prime Western blotting detection reagent (GE Healthcare). Western blots were imaged using the “lowest” sensitivity setting of the “Chemi” feature of an Azure Biosystems C400 imager. Uncropped immunoblots are available in Supplementary Figure 1.