One-step 10(4)-fold purification of transformed glucocorticoid receptor. Method for purifying receptors associated with Mr ca. 90,000 heat-shock protein

J Chromatogr. 1990 May 25;508(1):97-107. doi: 10.1016/s0021-9673(00)91243-3.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anion Exchange Resins
  • Antibodies, Monoclonal / immunology
  • Chromatography, Ion Exchange / methods
  • DNA / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Heat-Shock Proteins / isolation & purification*
  • Immunoblotting
  • Liver / analysis
  • Liver / ultrastructure
  • Molecular Weight
  • Rabbits
  • Receptors, Glucocorticoid / immunology
  • Receptors, Glucocorticoid / isolation & purification*
  • Receptors, Glucocorticoid / metabolism

Substances

  • Anion Exchange Resins
  • Antibodies, Monoclonal
  • Heat-Shock Proteins
  • Receptors, Glucocorticoid
  • DNA