Fig. 5

Comparison of the BRCA1-L protein binding to supercoiled and linear DNA by magnetic immunoprecipitation. a 0.3 μg superhelical pCFNO (lanes 3–5) and increased concentration of linear pCFNO/XhoI (0.3 μg—lane 3, 0.6 μg—lane 4, 1.2 μg—lane 5) were incubated with BRCA1-L protein (protein:DNA molar ratio 20:1) in binding buffer (5 mM Tris, 2 mM dithiothreitol 50 mM KCl, 0.01 % Triton X-100) with magnetic beads modified by the addition of BRCA1 monoclonal antibodies. BRCA1-DNA complexes were disrupted in a final step by incubation with 0.5 % SDS at 65 °C and then electrophoresed on 1 % agarose gel at 120 V and room temperature for 45 min. Lanes 1 and 2 contain control DNA of superhelical pCFNO and linear pCFNO/XhoI, respectively. b Graph representation of the BRCA1-L magnetic immunoprecipitation with different DNA targets. Ratio of the supercoiled and linear DNA before precipitation was 1:1. Densitometry of the DNA bands after immunoprecipitation with the BRCA1-L protein is shown in bar. c Scheme of the immunoprecipitation followed by DNA detection on magnetic beads