Monoclonal goat α rabbit hrp

For the in vivo crosslinking, cultures expressing different variants of TlpA-GFP were cultivated to an OD₆₀₀ of 0.5, rapidly chilled on wet ice, washed with and resuspended in ice cold PBS, and subsequently flash-frozen in liquid N₂. The cell suspensions were crosslinked with a tri-functional maleimide crosslinker tris(2-maleimidoethyl) amine (Pierce) at a concentration of 2 mg ml⁻¹ (10-min incubation at 30 °C under shaking). The crosslinking was subsequently blocked by the addition of 20 mM cysteine. Samples for SDS–polyacrylamide gel electrophoresis and western blotting were prepared with 10-min incubation with lysozyme at 30 °C, followed by brief sonication and dilution in SDS gel-loading buffer. SDS–polyacrylamide gel electrophoresis was performed with Novex NuPAGE 4–12% Bis-Tris Midi Gels (Life Technologies), western Blotting with polyvinylidene difluoride membrane (GE Healthcare), and detection of bound antibodies with ECL 2 Western Blotting Substrate (Pierce). The used antibodies were polyclonal rabbit α-GFP_6His (1:3,000, laboratory stock) and monoclonal goat α-rabbit HRP (1:10,000, Sigma).