Gel-shift assays of putative new HspR targets: SCO5639 and SCO4410. HspR-binding at the SCO5639 and SCO4410 promoter regions. Oligonucleotide pairs are detailed in Materials and methods. Protein extract from E. coli over-expressing hspR was incubated with 200 fmol biotinylated DNA fragment without competitor DNA (lanes 1-3) with, respectively, 4, 6 and 12 μg cell extract. In the lane marked 'C1' in the SCO5639 gel shift, 4 μg cell extract and 200-fold molar excess of specific competitor DNA were loaded. In lanes C1-C3 in the SCO4410 gel shift, 4, 6 and 12 μg cell extract were loaded, respectively, together with 200-fold molar excess of specific competitor DNA. Lane F shows unbound DNA (no added protein). Arrows indicate positions of bound (upper arrows) and unbound double-stranded DNA target.
Bucca et al. Genome Biology 2009 10:R5 doi:10.1186/gb-2009-10-1-r5