Sanguinarine, Putative Anticancer Therapeutic, Interacts with Chromatin, Modulates Epigenetic Modifications and Transcription in Context of Chromatin

Preparation of Chromatin and Mononucleosome from Rat Liver Purified nuclei were digested with micrococcal nuclease for the preparation of chromatin. Prolonged digestion of the nuclei with micrococcal nuclease was used to prepare mononucleosome with a DNA length of 146 base pairs. Selective solubilization of mononucleosome was done by the acidification (NaH2PO4 1.78% w/v) of the supernatant. The supernatant thus obtained was dialyzed against 0.01M Tris-HCl, pH 7.5, containing 0.01M EDTA and 0.002M PMSF. Final purification of mononucleosome was achieved by gel filtration using 25 cm x 1 cm sephacryl S-200 column. Purity of the chromatin and mononucleosome was checked by identification of histones in SDS-PAGE with commercially available sample of histones. The purity of the nucleosomal DNA was checked by agarose gel electrophoresis with appropriate markers. Concentrations of chromatin and mononucleosome are expressed in terms of DNA base. Chromosomal DNA was extracted from the chromatin by repeated phenol/chloroform for quantitative estimation of base.