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. 2009 May 1;191(13):4341–4352. doi: 10.1128/JB.00243-09

FIG. 4.

FIG. 4.

Determination of the affinity of P-PrrA for PrrA site 2 in the RSP3361 gene. (Top panels) Autoradiograms of the EMSA experiments. (Bottom panels) Quantitation. (A) Lane 1 contains a labeled 191-bp fragment containing the RSP3361 PrrA site 2 only. A calculated Kapp of 1.3 × 10−7 M corresponds to 50% occupancy, or 0.5 fractional saturation. (B) Approximately 0.35 pmol of the labeled double-stranded 191-bp fragment containing the RSP3361 PrrA site 2 was incubated with ∼0.57 μmol of P-PrrA, equivalent to the amount of P-PrrA used in lane 7 in panel A (top). In addition, increasing amounts of the double-stranded unlabeled 191-bp fragment containing the RSP3361 PrrA site 2 were added as specific competitor DNA. The competitor DNA amounts added are shown, and they constitute 1× (lane 3), 2× (lane 4), 5× (lane 5), 10× (lane 6), 20× (lane 7), and 50× (lane 8), relative to labeled DNA. Lane 1 contains a labeled 191-bp fragment containing the RSP3361 PrrA site 2 only, and lane 2 contains the labeled fragment and P-PrrA. A Ki value of 2.54 × 10−12 M indicates 50% displacement of P-PrrA, and it corresponds to ∼7.8×, relative to 1× the labeled DNA fragment.