Trans blot turbo mini 0.2 μm pvdf membrane

500 μl of an OD₆₀₀ = 0.7 culture of exponentially growing S. Typhimurium cells, induced with 0.2% arabinose at OD₆₀₀ = 0.3, were harvested by centrifugation at 4°C for 5 min at 21000 x g. Cells were re-suspended in 100 μl of sample buffer (50mM Tris pH 6.8, 1% SDS (w/v), 1% Triton X-100, 10% Glycerol, 0.2% bromophenol blue) and boiled for 5min at 95°C. 150mM DTT was then added to each sample followed by the pelleting of membrane debris and undigested genomic DNA at 21000 x g for 5 min before the supernatant were separated on a Mini-PROTEAN TGX gel (Biorad, USA). PageRuler Prestained Protein Ladder (Thermo Scientific, USA) was used as size marker. Proteins were then transferred to a Trans-Blot Turbo Mini 0.2 μm PVDF membrane (BioRad, USA) using the Trans-Blot Turbo system (Biorad, USA). RhsI^main-HA and RhsI^oprhan-HA proteins were detected using anti-HA antibody (11867423001, Sigma-Aldrich, Germany). Equal loading was confirmed by probing for RNA polymerase β-subunit using an anti-RNAP β-subunit antibody (ab191598, Abcam, United Kingdom). Secondary antibody towards anti-HA antibodies was anti-rat IgG coupled to HRP (GENA935, Sigma-Aldrich, Germany) and secondary antibody towards anti-RNAP antibodies was anti-rabbit IgG coupled to HRP (A1949, Sigma-Aldrich, Germany). Bands were visualized with Clarity Western ECL Substrate (Biorad, USA) and a ChemiDoc MP system (Biorad, USA).

Samples from solubilization and purification
steps were mixed with 1× NuPAGE LDS sample buffer and 1×
NuPAGE sample reducing agent. The mixture was heated at 50 °C
for 10 min. SDS-polyacrylamide gel electrophoresis was performed using
NuPAGE 4–12%, Bis-Tris gels. The gel was then transferred onto
a Trans-Blot Turbo Mini 0.2 μm PVDF membrane (Bio-Rad). The
membrane was blocked with 5% BSA in TBS-T (TBS + 0.1% Tween-20) for
1 h. Subsequently, the membrane was incubated with rabbit anti-V5
antibody (1:1000, Cell Signaling Technologies, D3H8Q) for 2 h at RT.
Antibody binding was detected using IRDye 680RD-conjugated Goat Anti-Rabbit
IgG H&L preabsorbed (1:10,000, Abcam, ab216777) for 1 h at RT.
The protein bands were visualized using the Odyssey CLx Imaging System
and quantified using ImageStudio software (Li-Cor).