A simple method for the purification of murine corticosteroid binding globulin: characterization of a monomer.
A fast and reproducible two-step method with high resolution was developed for purification of murine corticosteroid-binding globulin (CBG). The first step was liquid chromatography on a Sephacryl-S-200 column, and the CBG-containing residual was subsequently chromatographed by fast protein liquid chromatography (FPLC). This enabled us to quickly obtain a highly purified protein and the apparently isolated CBG was tested for its purity by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (SDS-PAGE) and sedimentation equilibrium centrifugation. The CBG concentration in pregnant mouse serum was estimated to 0.78 g/l (1.5% of the total protein). The monomeric organization of the protein was demonstrated by mercaptoethanol treatment. No NH2-terminal amino acid could be detected, probably owing to a blocked amino acid. The mol. wt (Mr) of murine CBG was determined to be 52,000 and the sedimentation constant S20 degrees, w to 3.9 S by analytical ultracentrifugation. The protein showed 5 bands when subjected to isoelectric focusing: 3 bands with apparent isoelectric points (pI) between pH 3.15-3.25 and two between pH 3.40-3.50.
Nyberg
L
L
Department of Clinical Chemistry, College of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden.
Jones
I
I
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007525:Isoelectric Focusing; D051379:Mice; D008970:Molecular Weight; D011247:Pregnancy; D014156:Transcortin / Q000302:isolation & purification*; D014461:Ultracentrifugation
D000596:Amino Acids; D002241:Carbohydrates; D014156:Transcortin