Electrophoretic Mobility Shift Assay for DNA Binding

To assess DNA binding of Rv0500A, promoter regions for rv2390c (full length—704 bp; 5’ section—403 bp; 3’ section—301 bp) and kdpF (754 bp) were amplified using IRDye 700 labeled primers (Integrated DNA Technologies) and the PCR products purified using a QIAquick PCR purification kit (Qiagen). Indicated amounts of purified His-Rv0500A were mixed with no more than 40 fmoles of DNA in EMSA buffer (20 mM Tris-HCl, pH 8, 50 mM KCl, 2 mM MgCl₂, 5% glycerol, 0.5 mM EDTA, 1 mM DTT, 0.05% Nonidet P-40, 25 μg/ml salmon sperm DNA [65 (link)]) in 11 μl final volume reactions. After incubation at room temperature for 20 minutes, the reactions were run on a non-denaturing 8% Tris-glycine gel in HEPES-imidazole buffer (35 mM HEPES, 43 mM imidazole). The gel was then imaged using the 700 nm channel of an Odyssey CLx imaging system (LI-COR).