Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection

Figure 6

Supershift assay with inclusion of antibody to BSA binding in gel retardation assays. A polyclonal antibody to BSA was tested for binding to BSA in the reaction complex and analyzed by EMSA. The negative control previously shown not to bind to BSA was used as well as I1-5, the BSA aptamer. For the negative control, no binding to BSA was seen at 100 μM or 500 μM BSA (lane 2 and 3). With addition of 4 μg of the antibody, there was some nonspecific binding, as seen by the faint band, by the control sequence (lane 4). However, with I1-5 there was binding to 500 μM BSA (lane 7), but this binding was disrupted when the antibody was incubated with BSA prior to the EMSA (lane 8). As stated previously, some of the radiolabeled DNA formed aggregates incapable of entering the gel, giving rise to the bands at the top of the lanes. A slight leak occurred during gelling such that the top of lane 8 appears disjointed due to the shape of the gel.