Purification of melanoma-associated oncofetal antigen gp87 from spent medium and cell homogenate of cultured human melanoma cells.
A melanoma-associated glycoprotein with an apparent molecular weight of 87,000 daltons (gp87) defined by the monoclonal antibody 140.240 has been purified from the spent medium and from cell homogenates of cultured human melanoma cells through a three-step purification procedure. The procedure involves a DEAE-Sephadex A-25 ion-exchange column, SephacrylS-200 gel filtration and antibody-Sepharose-4B-affinity chromatography. By this approach we achieved a 1205-fold increase in specific activity with a 42% antigen recovery from spent medium concentrate, and a 3366-fold increase in specific activity with a 29% antigen recovery from the cell homogenate. The antigen was purified from these two sources of starting material with a high degree of purity as visualized in a single protein band in SDS-polyacrylamide gel electrophoresis. Thus these procedures described offer an efficient approach to the purification of gp87 molecules for further biochemical studies.
D000951:Antigens, Neoplasm / Q000302:isolation & purification*; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification; D006801:Humans; D008545:Melanoma / Q000276:immunology*
D000951:Antigens, Neoplasm; D003470:Culture Media; D006023:Glycoproteins; C031192:oncofetal antigens