Anti his6 antibody

Purified His₆–GFP–Rad55 and FLAG–Rad57 complex (150 nM) was incubated with 150 nM His₆–Srs2 in buffer P (25 mM Tris–HCl [pH 7.5], 10 mM MgCl₂, 50 mM NaCl, 2.5 mM ATP, 1 mM DTT, 10% glycerol and 0.05% NP–40) for 1 h at 25°C. Equilibrated anti–FLAG beads (Sigma, Cat. No. A2220) were added to the mixture and incubated for 1 h. The beads and supernatant were separated by centrifugation, and the beads washed twice with binding buffer P. The protein complexes were eluted by boiling at 95°C for 5 min in SDS–PAGE loading buffer, resolved by a 10% SDS–PAGE gel, and the protein bands were visualized by immunoblotting with anti–His₆ antibody (Thermofisher, Cat No. MA1–21315–A488).

+toxI toxN-His₆ cells were grown at 30 °C in M9-glucose to OD₆₀₀ = 0.3 and infected with phage at MOI = 5. At every timepoint of interest, 1 mL of cells was pelleted and flash-frozen. Pellets were then resuspended at OD₆₀₀ = 15 (based on the OD₆₀₀ of the culture prior to infection) in 2x Laemmi loading dye supplemented with 20 mM DTT. As a loading control following T4 infection, His₆-FolC was added to each resuspended cell pellet at a final concentration of 0.2 fM. Samples were then analyzed by 4–20% SDS-PAGE and transferred to a PVDF membrane. To visualize proteins, anti-His₆ antibody (ThermoFisher) was used at a final concentration of 1:5000, and then SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher) was used to develop the blots. Blots were then imaged with a FluorChem R Imager (ProteinSimple). As a loading control following rifampicin treatment, blots were then re-probed with anti-RpoA antibody at a final concentration of 1:5000. Blots shown are representative of two independent replicates.

Cells were harvested by centrifugation and flash frozen in liquid nitrogen. Pellets were resuspended in 1x Laemmli buffer and analyzed by SDS-PAGE. Anti-MqsRA antibody was generated using His₆-MqsRA complex (Covance) and used unpurified at 1:3000. Anti-His₆ antibody was used at 1:5000 (Thermo Fisher); and purified anti-Lon antibody was used at 1:5000 (kind gift from T. Baker and R. Sauer). SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher) was used to develop the blots, which were visualized with a FluorChem R Imager (ProteinSimple). Because high amounts of lysate were loaded onto gels to allow for antitoxin detection, typical loading controls were outside the linear range. Loading normalization was instead done by Coomassie staining membranes after Western blotting. All Western blot data presented are the average of three independent biological replicates. As for the pulse-chase experiments, samples were collected and processed for 90 and 120 minutes; however, we only present data up to 60 minutes for the reasons described above.