Characterizing Transcriptional Regulators with EMSAs

Electrophoretic mobility shift assays (EMSAs) were conducted as previously described (MacGilvary et al., 2019 (link)). Promoter regions of ftsK (771 bp), ftsW (717 bp), erp (746 bp), ripA (694 bp), and rv2390c (704 bp) were amplified using IRDye 700 labeled primers (Integrated DNA Technologies). PCR products were purified using a QIAquick PCR purification kit (Qiagen). Recombinantly purified Rv0500A (WT or M39A/T40A mutant) was mixed with no more than 30 fmol of DNA for 20 minutes at room temperature as previously described (MacGilvary et al., 2019 (link)). For competition EMSAs with the unlabeled probe, 200 fmol of unlabeled DNA was included in the mix with 1 fmol of labeled DNA and 0.1 μM of recombinantly purified WT Rv0500A. Samples were then loaded onto a non-denaturing Tris-glycine gel (5%) in HEPES-imidazole buffer (35 mM HEPES, 43 mM imidazole) and imaged after running using the 700 nm channel of a LI-COR Odyssey CLx imaging system.