Rabbit anti btug2

A B. thetaiotaomicron strain expressing an HA-epitope tagged allele of the periplasmic protein SusA (Shipman et al., 1999 (link)) was grown to OD600 ~0.8 in minimal media with methionine, pelleted and washed in 1x cOmplete EDTA-free protease-inhibitor cocktail (Roche, Basel, Switzerland) before being pelleted and stored at −80 ˚C. Pellets were thawed and resuspended in PBS with proteinase K (0, 10, 50 or 100 µg/mL; AmericanBio, Natick, MA, USA), and incubated at 37 ˚C aerobically under continuous agitation (250 rpm) for 8 hr. Cells were then pelleted and washed 3 times in 1x cOmplete EDTA-free protease-inhibitor cocktail, pelleted and stored at −80 ˚C. Thawed cells were lysed using BugBuster reagent (Millipore Sigma, Burlington, MA, USA), 20 µg of clarified protein lysate was loaded onto an SDS-PAGE gel, transferred to a PVDF membrane and probed with rabbit anti-BtuG2 and rabbit anti-HA (Santa Cruz Biotechnology, Dallas, TX, USA).

B. thetaiotaomicron cultures were grown to OD600 ~0.6 in minimal media with methionine. Cells were pelleted (~3000 x g for 15 min at 4˚C) and supernatant was filtered through 0.2 µm filter and stored temporarily on ice. Pellets were resuspended in breakage buffer (50 mM Tris pH 8, 5 mM EDTA, 2 mM PMSF, 10% glycerol), lysed at 4˚C by sonication (40 Amps; 15 s ‘on’ and 30 s ‘off’; 3 min total), and clarified lysates were ultracentrifuged at 100,000 x g for 1 hr at 4˚C to separate membranes (insoluble) from cytoplasm/periplasm (soluble) fractions. Membrane fractions were resuspended in 250 µl of breakage buffer, while the cytoplasm/periplasm fraction was concentrated by centrifugal filtration (30K; Millipore Sigma, Burlington, MA, USA) to 250 µl. Membrane and cytoplasm/periplasm fractions were temporarily stored on ice. Filtered supernatant was utracentrifuged at ~100,000 x g for 1 hr at 4˚C to remove outer membrane vesicles. The soluble fraction was then concentrated by centrifugal filtration to 250 µl. 20 µl each of membrane, cytoplasm/periplasm, and supernatant fractions were loaded onto SDS-PAGE gels and analyzed by Western blot. PVDF membranes were probed with rabbit anti-BtuG2 and mouse anti-RpoB (Santa Cruz Biotechnology, Dallas, TX, USA) as a cytoplasmic control.