Preparation method of highly-purified antibodies from antisera by using polyvinylidene difluoride membrane. Application to fatty acid binding protein in rat liver.
A simple method for preparation of highly-purified antibodies from antiserum was developed by using antigen-blotted polyvinylidene difluoride transfer membrane as an affinity matrix: The antigen was purified by SDS-polyacrylamide slabgel electrophoresis. Purified antibody preparations, which had been eluted from the Gly-HCl buffer (pH 2.5)-pretreated affinity matrix, by treatment with 1,4-dioxane, were more effective to obtain specifically stained band in immunoblot analysis than antiserum or immunoglobulin fractions purified by already reported methods. Antigen-blotted membranes were repeatedly available without remarkable release of antigen or decrease of antigenicity. This method was also applied to the examination of cross-reacting proteins with rat liver fatty acid binding protein in the cytosol fraction.
Nishino
H
H
Department of Biochemistry, Hokkaido University School of Medicine, Sapporo, Japan.
Ishibashi
T
T
Nakamura
H
H
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000918:Antibody Specificity; D000941:Antigens / Q000302:isolation & purification; D002352:Carrier Proteins / Q000032:analysis / Q000276:immunology*; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000378:metabolism; D004146:Dioxanes; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005260:Female; D005998:Glycine; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting / Q000379:methods; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007202:Indicators and Reagents; D008099:Liver / Q000378:metabolism; D008567:Membranes, Artificial; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011145:Polyvinyls; D011817:Rabbits / Q000276:immunology; D051381:Rats; D012709:Serum Albumin / Q000032:analysis / Q000276:immunology*; D025521:Tumor Suppressor Proteins; D005465:Fluorocarbon Polymers
D000906:Antibodies; D000941:Antigens; D002352:Carrier Proteins; D004146:Dioxanes; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005227:Fatty Acids; D005998:Glycine; D007106:Immune Sera; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D008567:Membranes, Artificial; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D011145:Polyvinyls; D012709:Serum Albumin; D025521:Tumor Suppressor Proteins; C107608:FABP7 protein, human; C490174:Fabp7 protein, rat; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C025223:1,4-dioxane