982140.fig.004a
(a) EMSA
982140.fig.004b
(b) DNase I footprinting
Figure 4: DNA binding analysis. (a) EMSA.The radioactively labeled DNA fragment from the 504 bp upstream to the 40 bp downstream of hcp1 was incubated with increasing amounts of purified His-OpaR protein (lanes 1, 2, 3, 4, 5, 6, and 7 containing 0, 1.7, 2.0, 2.3, 2.6, 3.0, and 3.4 pmol, resp.) and then subjected to 4% (w/v) polyacrylamide gel electrophoresis. Shown also was the schematic representation of the EMSA design. (b) DNase I footprinting. Labeled coding or noncoding DNA probes were incubated with increasing amounts of purified His-OpaR (lanes 1, 2, 3, and 4 containing 0, 6, 12.5, 18.8, and 25 pmol, resp.) and subjected to DNase I footprinting assay. Lanes G, A, T, and C represented the Sanger sequencing reactions. The footprint regions were indicated with vertical bars. The negative or positive numbers indicated the nucleotide positions upstream or downstream of hcp1, respectively.