Maximizing the purification of the activated glucocorticoid receptor by DNA-cellulose chromatography.
With heat treatment (20 degrees C for 30 min), the glucocorticoid-receptor complex becomes 'activated' and undergoes an increase in affinity for DNA. A two-stage procedure was used to separate sequentially the rat liver glucocorticoid-receptor complex from proteins with high and low affinity for DNA. DNA-cellulose column chromatography of unheated cytosol resulted in the retention of DNA-binding proteins, but not the unactivated receptor complex. Heat treatment of the column eluate resulted in increased affinity of the receptor complex to DNA, and chromatography on DNA-cellulose then yielded receptor complex free from proteins with low affinity for DNA. Removal of DNA-binding proteins during the first chromatographic step was critically dependent on ionic conditions and the ratio of cytosol chromatographed to DNA-cellulose. A purification of 11000-fold (85% yield) was achieved by this procedure. The partially purified receptor complex was taken up by rat liver nuclei.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002482:Cellulose; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D008297:Male; D011485:Protein Binding; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification* / Q000378:metabolism; D011987:Receptors, Steroid / Q000302:isolation & purification*; D014221:Triamcinolone / Q000378:metabolism
D011965:Receptors, Glucocorticoid; D011987:Receptors, Steroid; D014221:Triamcinolone; D002482:Cellulose; D004247:DNA