To address whether FadR can bind to the V. cholerae plsB promoter region, gel shift assays were conducted as we recently reported (Feng & Cronan, 2009b (link), Feng & Cronan, 2010 (link), Feng & Cronan, 2011 ) with minor modifications. Two DNA probes were used, one of which is about 100 bp of a PCR product obtained by amplification with specific primers plsBvc_P-F plus plsBvc_P-R (Table 2). The second DNA probe containing the predicted FadR binding palindrome was generated by annealing two complementary oligonucleotides (plsBvc_FadR_BS-F plus plsBvc_FadR_BS-R, Table 2) by incubated at 95 °C in TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, pH 8.0) for 5 min followed by slow cooling to 25 °C. The probes were labeled with digoxigenin by terminal transferase incorporation of digoxigenin-ddUTP (Roche). The digoxigenin-labeled DNA probes (~1 pmol) were mixed with purified FadR (in appropriate concentrations) in the binding buffer (Roche) and incubated 15–20 min at room temperature. The DNA/protein mixtures were then analyzed by native PAGE (6% PAGE for the ~100 bp PCR probe, and 8% PAGE for the 39 bp synthetic probe). Contact blotting-aided gel transfer was conducted,followed by UV cross-linking (120 mJ for 180 s), 1 h of blocking of the nylon membrane in 50 ml blocking buffer, and 1 h of incubation with an anti-digoxigenin antibody solution (1:10,000) at room temperature. Subsequently the nylon membrane was washed 5 times (15 min each) and equilibrated with detection buffer for 15 min before 1 h of development of the luminescent reaction in CSPD working solution (Roche) at 37 °C. Finally the membrane was exposed to ECL film (Amersham) for signal capture.