Nuclear Protein-DNA Interaction Analysis

HEK 293T cells were cultured in large quantities, and the lysates underwent subcellular fractionation to obtain a nuclear pellet, which was then dialysed. The pBSK (+) duplex plasmid DNA was linearised with EcoRI and then co-incubated with nuclear extracts, in which genomic DNAs were fragmented into small pieces (<500 bp) by sonication, in reaction buffer at 37 °C for 1 h according to previously described methods^{22 (link),23 (link)} and then subjected to 0.7% agarose gel electrophoresis after deproteinisation (10 μg/μl proteinase K, 3% SDS, 50 mM EDTA and 100 mM Tris-HCL, pH 7.5, 90 min at 57 °C). The gel was then incubated with the highly sensitive nucleic acid dye GelRed (Sigma-Aldrich) at 1:10,000 dilution in water at room temperature for 15 min and then de-stained in water for ~1 h. The Quantification of the DNA bands was performed using a phosphorimager (Analytik Jena AG).