FadR-DNA Interaction Assay Protocol

Electrophoretic mobility shift assays were performed to address interaction between FadR and series of taget DNA probes (e.g., fadD and fadL (Feng & Cronan, 2012 (link))) essentially as previously reported (Feng & Cronan, 2011b (link), Feng & Cronan, 2011a (link), Feng & Cronan, 2009b (link)). With an exception of fadM probe (PCR products) (Feng & Cronan, 2009b (link)), all of the FadR-recognizable probes were prepared by annealing two complementary primers (Table 2) by incubation in TEN buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, 100 mmol/L NaCl, pH 8.0) at 95°C for 5 min followed by slow cooling to 25°C and then digoxigenin (DIG) labeling by terminal transferase with DIG-ddUTP (Roche). The DIG-labeled DNA probes (either 0.1 pmol or 0.2 pmol) were incubated with either DNA binding protein in binding buffer (Roche) for 15 min at room temperature and then analyzed by native PAGE (7–7.5% PAGE for all probes).