Purification of a brain-specific astroglial protein by immunoaffinity chromatography

An immunoaffinity chromatography procedure for the isolation of bovine glial fibrillary acidic (GFA) protein is described. Degraded GFA protein isolated by hydroxyapatite chromatography from human spinal cord was used to prepare the antiserum. The immunoglogulin G fraction of the antiserum was covalently linked to CNBr-activated Sepharose, and columns of the immuno-affinity gel were used to adsorb bovine GFA protein from brain extracts. Elution was accomplished with a solution of 1 m acetic acid, 5 m urea, 0.8 m sodium chloride, pH 2.5. The yield of about 0.5 mg of highly purified protein/g of cerebral white matter could be increased to 1.5 mg/g of tissue by lowering the ionic strength of the extracting buffer from 50 mm to 1 mm sodium phosphate. Isolation in the presence of EDTA prevented the formation of an oxidation product migrating as a dimer of the monomeric species on SDS-polyacrylamide gel electrophoresis.