Journal: Molecular Microbiology

Article Title: Processing by rhomboid protease is required for Providencia stuartii TatA to interact with TatC and to form functional homo-oligomeric complexes

doi: 10.1111/j.1365-2958.2012.08080.x

Figure Lengend Snippet: E. coli TatA co-purifies with TatC in the absence of TatB. TatA and TatC his were co-produced in E. coli strain DADE (Δ tatABCD Δ tatE ). Membrane proteins were solubilized in digitonin. His-tagged TatC and associated TatA was purified using nickel IMAC chromatography and visualized by Coomassie Blue staining after SDS-PAGE (inset). Purified TatAC his complexes were subjected to gel filtration chromatography on a Superose 6 column and eluted as a broad peak (main image). Beta-amylase (200 kDa), apoferritin (443 kDa) and thyroglobulin (669 kDa) were used for molecular weight calibration (arrows). TatA and TatC were detected in the gel filtration fractions (indicated by lines) by Western blotting. All analysed fractions contained both TatA and TatC (bottom).

Article Snippet: Nine hundred microlitres of solubilized membrane fraction was mixed with 100 μl of Profinity IMAC Ni-Charged resin (Bio-Rad) and gently pelleted by centrifugation at 400 g for 1 min at 4°C.

Techniques: Produced, Membrane, Purification, Chromatography, Staining, SDS Page, Filtration, Molecular Weight, Western Blot