Anti his hrp

The purification of TatC‐his and SufI‐his‐containing complexes produced in strain DADE/pRep4 from the pUNITAT2 and pFAT75ΔASufIhis plasmids, respectively, or of chromosomally encoded TatC‐his from strain MC75CH was performed as described in Fritsch et al. (2012) with the following modifications: Buffer B (20 mM MOPS pH 7.2, 200 mM NaCl) was used as resuspension buffer; membrane protein was solubilised using 1% digitonin in Buffer B supplemented with 50 mM imidazole (25 mM imidazole for chromosomally encoded TatC‐his); Buffer B containing 50 mM imidazole (25 mM imidazole for chromosomally encoded TatC‐his) and 0.1% digitonin was used as wash buffer; and Buffer B containing 10 mM EDTA and 0.1% digitonin was used as elution buffer (as protein samples in high concentrations of imidazole run poorly for BN‐PAGE).
Samples from each step of the purification were diluted in 1× Lammli buffer, separated by SDS–PAGE (12% acrylamide) transferred to nitrocellulose membrane and immunoblotted with anti‐his‐HRP (Abcam, 1/50,000 dilution to detect TatC‐his and 1/20,000 to detect SufI‐his), anti‐TatC (1/10,000, to detect untagged TatC bound to SufI‐his) anti‐TatB (1/1000) and anti‐TatA [raised in rabbits and used at 1/10,000 dilution (Sargent et al., 2001)].
Goat anti‐rabbit and anti‐mouse HRP conjugated antibodies (BioRad) were used at 1/10,000 dilution.

Briefly, protein bands separated on SDS-PAGE were transferred to a methanol-activated polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in 5% skim milk in PBST for 1 h. Subsequently, the membrane was incubated with 10 μg mL⁻¹ primary antibody or Fab followed by incubation with goat-anti-human IgG-HRP (Thermo Fisher Scientific, cat. #31413, diluted 10,000×) or anti-his-HRP (Abcam, cat. #ab18184, diluted 1000×). Three washes for 5 min in PBST were conducted after each incubation step. WesternBright ECL (Advansta) was added for protein visualization.