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. Author manuscript; available in PMC: 2016 Apr 12.
Published in final edited form as: Mol Microbiol. 2013 Mar 14;88(1):105–124. doi: 10.1111/mmi.12171

Fig. 5.

Fig. 5

BadR binds rpoS promoters. Schematic diagram illustrating the two regions upstream of rpoS used for the EMSAs. RpoS 1 promoter (PrpoS 1(813088-813282)) includes the RpoS start codon (boxed) and BosR binding site 3 (BS3 -Underlined). RpoS2 promoter (PrpoS 2(813258-813471)) includes the RpoN binding site (bold), both the BosR binding sites 1 and 2 (BS1, BS2 -Underlined), and the rpoS transcriptional start site (bolded with asterisk) (A). 5′ biotin-labeled promoters (2 nmols) were mixed with various amounts of purified BadR(734081-735289) (130, 200, 270 pmols) in a 20μl binding reaction. Some reactions were incubated with unlabeled promoters (200-fold molar excess) for competition reactions or a N′ terminal HTH deficient BadR(734225-735289) (BadR Δ HTH) (B). The binding reactions were incubated at room temperature for 20 minutes, run on a 6% polyacrylamide gel, and transferred onto a positively charged Nylon membrane. After transfer the membrane was cross-linked by UV irradiation, blocked, incubated with streptavidin-horseradish peroxidase conjugate and luminol enhancer substrate solution allowed visualization of labeled DNA by exposure to X-ray film.