Pd spintrap g 25desalting column

These experiments were performed as described in our previous study (59 (link), 60 (link)). Briefly, purified RavS or RavS^H503A was incubated with c-di-GMP or the purified proteins as indicated in the presence of 20 μM [γ-³²P]ATP (Perkin-Elmer, USA) in reaction buffer (50 mM Tris-HCl [pH 7.8], 25 mM NaCl, 25 mM KCl, 5 mM MgCl₂, 2 mM DTT) at 28°C for the indicated times. In RavS-RavR phosphoryl transfer assays, purified RavS or RavS^H503A were incubated with 20 μM [γ-³²P]ATP (Perkin-Elmer, USA) in reaction buffer for the indicated times at 28°C. The remaining ATP was depleted by using a PD-Spintrap G-25desalting column (GE, New York, NY) before the next step. c-di-GMP, wherever necessary, was added to the reaction system simultaneously with the other proteins. The reactions were terminated by adding 5× SDS-PAGE loading buffer. Samples were loaded into the gels for PAGE and then exposed to a phosphor screen (GE Healthcare) to detect autoradiographic signals by a Typhoon FLA7000 (GE Healthcare).

Assays were performed as described in our previous study (19 (link), 23 (link)). The probe, the promoter region of fliD, was amplified by PCR and labeled by [γ-³²P]ATP using T4 polynucleotide kinase. The labeled probes and purified proteins were incubated in reaction buffer containing 10 mM Tris (pH 7.0), 50 mM KCl, 1 mM dithiothreitol (DTT), 2.5% glycerol, 5 μM MgCl₂, 50 ng L⁻¹ poly(dI:dC), 0.05% NP-40, and 10 mM EDTA for 50 min at room temperature. ATP used to phosphorylate RavS was depleted using a PD-Spintrap G-25 desalting column (GE, New York, NY) before the addition of FsnR. Reactions were stopped by the addition of loading buffer (0.25% bromophenol blue, 40% sucrose), and samples were subjected to polyacrylamide gel electrophoresis (PAGE) in a 5% native gel and then exposed to a phosphor screen (GE Healthcare) for detection of the autoradiographic signals by a Typhoon FLA7000 (GE Healthcare). Unlabeled probes were used as the cold probes in the competition experiments.