Extraction of a murine tumor-specific transplantation antigen with 1-butanol. I. Partial purification by isoelectric focusing.
Treatment of whole viable MCA-F murine sarcoma cells from syngeneic female C3H/HeJ mice with a single-phase solution of 2.5% (vol/vol) 1-butanol yielded a crude membrane protein extract without loss of cell viability. 1-Butanol-extracted cells were capable of in vitro proliferation. Variations in extraction conditions significantly influenced not only protein yield but also viability. The crude butanol extract (CBE) contained a tumor-specific transplantation antigen (TSTA) more potent than that obtained by 3-M KCl extraction of MCA-F cells: a specific activity of 40 TSTA U/mg protein in the former and 2 U/mg in the latter. The antigen was specific and protected host against challenge with MCA-F cells but not the antigenically different MCA-D cells. The growth of MCA-F cells was not reduced by pretreatment with equivalent doses of CBE obtained from MCA-D cells. Partial purification of the antigenic activity by preparative isoelectric focusing revealed that most activity focused in the pH 6.4 fraction, which increased the specific activity to 1,000 TSTA U/mg protein. Thus 1-butanol was an effective agent for the extraction of certain membrane polypeptides, including an immunogenic TSTA, from whole cells.
D000818:Animals; D005260:Female; D005354:Fibrosarcoma / Q000276:immunology* / Q000473:pathology; D005720:Gamma Rays; D006649:Histocompatibility Antigens / Q000008:administration & dosage / Q000276:immunology / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007114:Immunization; D007525:Isoelectric Focusing; D051379:Mice; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000276:immunology; D009994:Osmolar Concentration; D013696:Temperature; D014185:Transplantation, Isogeneic
D006649:Histocompatibility Antigens