Nativepage bis tris protein gels

Samples consisting of either solubilized membranes or purified membrane complexes were mixed with 5% G-250 sample additive (Invitrogen), to a final concentration of about 0.2%, and run on 3–12% NativePage Bis-Tris Protein Gels (Invitrogen) according to manufacturer specifications. Gels were either stained with Coomasie R-250 or transferred to PVDF membranes and stained with appropriate antibodies (Supplementary Fig. 1). Antisera against EccB₅, used in Extended Data Fig. 1b, was raised against the synthetic peptide CLPMDMSPAELVVPK and has previously been described^{16 (link)}. Polyclonal rabbit antisera against the peptide was raised in rabbits by Innovagen using Stimune (Prionix) as adjuvants. The antibody was used as a 1:5,000 dilution. Blots were visualized on a ChemoStar TouchMotionCor2 using ChemoStarTS.

For BN-PAGE analysis of membrane complexes, cell envelopes at a concentration of 3 mg/mL were solubilized with 0.25% DDM as stated above. The resulting supernatant samples were run on 3–12% native PAGE Bis-Tris Protein Gels (Invitrogen) with the addition of native PAGE 5% G-250 Sample Additive (Invitrogen) according to the manufacturer’s specifications. Gels were blotted to polyvinylidene difluoride (PVDF) membrane and stained with appropriate antibodies, as stated above.