Immunoaffinity chromatography on antibodies immobilized on nitrocellulose powder.
A method is described for the use of nitrocellulose powder as a solid phase in a chromatographic procedure, for the immunoaffinity isolation of proteins. Two different immunoglobulins (Igs), anti-Datura innoxia lectin and anti-tyrosinase, were coupled to particulate nitrocellulose. A single step was then needed for purification to homogeneity of both D. innoxia lectin and mouse tyrosinase. Chaotropic and acidic agents proved to be effective in eluting antigens from Ig-nitrocellulose columns. The binding capacity of particulate nitrocellulose was around 3 mg Ig per milliliter of nitrocellulose, while the purification yields of the two proteins investigated under various eluting conditions were higher than 75%. The applicability of this method in the identification of metabolically labeled proteins in crude extracts is also demonstrated. Purification of proteins by affinity chromatography on their specific Igs linked to nitrocellulose matrices could be performed in both batch and column. The major advantages of this new method for purification of proteins are its rapidity, the reusability of the affinity matrices, and the high yields of purified protein obtained. The method could be seen as an alternative to the widely used immunoprecipitation technique.
D000818:Animals; D000906:Antibodies; D001345:Autoradiography; D002686:Chitin; D002846:Chromatography, Affinity / Q000379:methods*; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007074:Immunoglobulin G; D007136:Immunoglobulins / Q000276:immunology / Q000302:isolation & purification*; D008546:Melanoma, Experimental; D051379:Mice; D014442:Monophenol Monooxygenase / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits / Q000276:immunology; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured
D000906:Antibodies; D007074:Immunoglobulin G; D007136:Immunoglobulins; D013462:Sulfur Radioisotopes; D002686:Chitin; D003101:Collodion; D014442:Monophenol Monooxygenase