Purification of Gc-2 globulin from human serum by displacement chromatography: a model for the isolation of marker proteins identified by two-dimensional electrophoresis.
A protein that was initially known only as a minor spot in two-dimensional electrophoresis patterns of serum obtained from certain psoriasis patients, particularly those with a pustular component to their disease, has been purified by two stages of ion-exchange displacement chromatography on DEAE-Sephacel at different pH levels, followed by elution chromatography on hydroxylapatite. The purification was followed by examining the column fractions directly by two-dimensional gel electrophoresis. The capacity of the displacement system, which utilized carboxymethyldextrans as displacers, was very high; 6 ml of dialyzed serum applied to a 7-ml column in the initial stage resulted in a very substantial enrichment of the target protein. The second displacement stage yielded a highly purified product, contaminated only by A-1 lipoprotein. The latter was removed by hydroxylapatite chromatography. The purified protein was subsequently identified as Gc-2 globulin, a vitamin D-binding protein, by immunological procedures. The results demonstrate the effectiveness of ion-exchange displacement chromatography in focusing resolving power on the relatively narrow range of affinities represented by the target protein and its immediate neighbors in a chromatogram, as well as the applicability of the system to the isolation of a protein known only by its position in a two-dimensional electrophoretic pattern.
Torres
A R
AR
Krueger
G G
GG
Peterson
E A
EA
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001797:Blood Protein Electrophoresis / Q000379:methods; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D017886:Durapatite; D006801:Humans; D006882:Hydroxyapatites; D008956:Models, Chemical; D011565:Psoriasis / Q000097:blood; D012710:Serum Albumin, Bovine / Q000032:analysis; D014809:Vitamin D-Binding Protein / Q000302:isolation & purification*
D000906:Antibodies; D006882:Hydroxyapatites; D014809:Vitamin D-Binding Protein; D012710:Serum Albumin, Bovine; D017886:Durapatite