Verification of AtoC in vitro binding to representative "atypical" targets. A) Sample gel retardation experiments illustrating His10-AtoC protein binding to DNA fragments atoD, metR-metE, acrD (promoter) and narG (gene encoding) regions. As described in Materials and Methods, following electrophoresis, gels were first stained with Gel-Red dye and then with Coomassie blue, and side-by-side photographs are shown. The lines above the Gel-Red stained lanes indicate the different probes combined with the indicated His10-AtoC (AtoC) quantities. Arrows indicate bands that were stained with both Gel-Red and Coomassie blue, corresponding to band-shifting caused by the AtoC binding. Lines indicate free proteins, not bound to corresponding DNA particles. B) Band shift assay of His10-AtoC with a biotinylated fragment of the upstream region of the atoDAEB operon. EMSAs were performed as described in Materials and Methods with 10 ng of biotinylated atoD. The addition of His10-AtoC (0.35 μM) and certain amounts of competitive, non-biotinylated atoD, as well as metR-metE, acrD and narG fragments is indicated. 2 μg of sonicated calf thymus competitive, non-specific DNA were added in each reaction.
Pilalis et al. BMC Genomics 2011 12:238 doi:10.1186/1471-2164-12-238