Novex nupage 10 bis tris gels

Two plasmids were used for co-expression in E. coli: a pET-TEV derivative encoding IrrE or IrrEΔGAF (both with N-terminal His-tag) and a pET22b derivative encoding DdrO (with C-terminal His-tag). E. coli BL21 (AI) cells freshly transformed with the two plasmids were grown at 37 °C overnight to saturation in 10 ml of LB medium containing kanamycin and ampicillin. This pre-culture was used to inoculate (start OD₆₀₀ 0.05) 100 ml of LB medium with the antibiotics and grown at 37 °C with aeration. At OD₆₀₀ of 0.6–0.7, IPTG (0.1 mM final concentration) and L-arabinose (0.2% final concentration) were added and the cells were further incubated at 37 °C for 3 h. Five hundred µl of induced cells were taken, centrifuged (10,000g, 2 min, 4 °C) and re-suspended in Novex NuPAGE LDS Sample Buffer (100 µl of sample buffer for 1 ml of culture at OD₆₀₀ 1), and heated for 10 min at 95 °C. Samples (20 μl per lane) were loaded on SDS-PAGE gels (Novex NuPAGE 10% Bis–Tris Gels; Invitrogen), and migrated in 1X NuPAGE MES SDS Running Buffer (Invitrogen) for 1 h at 130 V. In vitro cleavage control was performed as described previously^{14 (link)}. The proteins were visualized by staining with Imperial protein stain (Pierce).