Figure 1. In vitro phosphorylation of CpxR by acetyl∼P.

Wild type CpxR (CpxR_wt::His₆) was purified by metal affinity chromatography. Purified protein (1.25 µM) was incubated in sequentially increasing concentrations of acetyl∼P (AP). Samples were fractionated on a Manganese(II)-Phos-tag™ 12.5% acrylamide gel and then visualized by the SYPRO® Ruby staining method. Lanes: a, no addition of AP; b, 6.25 mM AP; c, 12.5 mM AP; d, 25 mM AP; e, 50 mM AP; f, 100 mM AP. Phosphorylated CpxR is indicated by a double asterisk and unphosphorylated CpxR by a single asterisk.