One-step 10(4)-fold purification of transformed glucocorticoid receptor. Method for purifying receptors associated with Mr ca. 90,000 heat-shock protein.
Chromatography of rabbit glucocorticoid-receptor complexes in the absence of sodium molybdate on a Mono Q anion-exchange column induces the transformation of the receptor and allows the resolution of the transformed and non-transformed molecular species. These abilities were used to design a new purification scheme for the glucocorticoid receptor from rabbit liver in its transformed state. Microgram amounts of receptor were obtained using this single-step procedure in less than 2 h. The purification yield was 50-60%. Immunoblot experiments showed that the glucocorticoid receptor was present as an Mr approximately 94,000 polypeptide in these preparations and represented 20-30% of the eluted proteins as determined by densitometric scanning analysis of silver-stained sodium dodecyl sulphate polyacrylamide gels. Finally, the purified receptor was able to interact quantitatively with bulk DNA.
Denis
M
M
Department of Medical Biochemistry, Nantes University Hospital, France.
Blanchardie
P
P
Orsonneau
J L
JL
Lustenberger
P
P
D000818:Animals; D000837:Anion Exchange Resins; D000911:Antibodies, Monoclonal / Q000276:immunology; D002852:Chromatography, Ion Exchange / Q000379:methods; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000302:isolation & purification*; D015151:Immunoblotting; D008099:Liver / Q000032:analysis / Q000648:ultrastructure; D008970:Molecular Weight; D011817:Rabbits; D011965:Receptors, Glucocorticoid / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D000837:Anion Exchange Resins; D000911:Antibodies, Monoclonal; D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D004247:DNA