Effects of estrogen on gene expression in the chick oviduct. Isolation and fractionation of chromatin non-histone proteins.
Non-histones isolated from hen oviduct chromatin have been fractionated by a variety of methods. Chromatin was dissociated in 2 M NaC1, 5 M Urea, 0.1% beta-mercaptoethanol and 0.01 M Tris - HC1, pH 8.3, and the DNA removed by ultracentrifugation. After desalting by gel filtration the chromatin proteins were separated into three distinct fractions by stepwise elution with 0.10 M NaC1, 0.25 M Na C1 and 15% guanidine - HC1 from Bio-Rex 70 columns. Fractions I and II contain only non-histones and Fraction III contains histones plus a small amount of non-histones. Further fractionation of the non-histones was achieved by ammonium sulfate precipitation and DEAE-cellulose chromatography for Fraction I and phosphocellulose chromatography and gel filtration on Bio-Gel A-15 m for Fraction II. The histone and non-histones present in Fraction III were separated by gel filtration on Bio-Gel A-0.5 m. All fractionation methods have been used preparatively with reasonable recoveries of protein (greater than or equal to 60%). The fractions have been characterized by acrylamide gel electrophoresis. The integrity of the histones was maintained during the fractionation procedure indicating that proteolytic degradation was unlikely to have occurred. There was no selective loss of chromatin proteins during the ultracentrifugation and desalting steps and the non-histones were separated into distinct fractions with enrichment of some species not apparent prior to fractionation of the chromatin proteins.
D000818:Animals; D002645:Chickens; D002843:Chromatin / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D008970:Molecular Weight; D010057:Oviducts / Q000378:metabolism*; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone