NceR Protein Binding Assay using FAM/HEX-labeled ompA Probe

A PCR fragment spanning the ompA promoter was amplified using the 6-carboxyfluorescein (FAM)- and 6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein (HEX)-labeled primers [FAM]ompAFL and [HEX]ompR. NceR protein binding to the labeled DNA probe and DNAse I digestion reactions were performed as described previously (44 (link)). Detection of the DNAse I digestion peaks was carried out in a 3730 capillary sequencer (Applied Biosystems, Waltham, MA, USA) by Azenta Life Sciences (Chelmsford, MA, USA), and the alignment of the corresponding electropherograms was generated using GeneMapper Software Version 4.0 (Applied Biosystems). Negative control reactions were done using bovine serum albumin (BSA) at the same mass concentration used for NceR. Phosphorylated MisR protein was prepared as previously described as a positive control (45 (link)). A DNA template was amplified with primers ompAFL and ompR to generate a sequence ladder for each strand as previously described (46 (link)). The final electropherograms of the sequencing reactions were horizontally aligned with those generated in the footprint using GeneMapper Software Version 4.0 (Applied Biosystems). As confirmation, the HEX/FAM-labeled DNA probe was subjected to Sanger sequencing using primer pompR.