Isolation of glial fibrillary acidic protein from bovine brain white matter and its purification by affinity chromatography on single-stranded DNA-cellulose.
Myelinated axons, which had been prepared from bovine brain white matter employing the flotation method, were extracted with Triton X-100 in a low ionic strength buffer containing 4 mM Mg2+. After delipidation of the detergent-resistant, residual material with chloroform-methanol, glial fibrillary acidic protein (GFAP) was solubilized with 6 M urea. It was enriched by ion exchange chromatography in the presence of 6 M urea on CM-Sepharose CL-6B at pH 5 and on DE52-cellulose at pH 7.6, respectively. The final purification was achieved by affinity chromatography on single-stranded DNA-cellulose in 6 M urea. Employing SDS-polyacrylamide gel electrophoresis, the molecular weight of the purified GFAP was determined to be 51,000. 2D-polyacrylamide gel electrophoresis revealed a major protein constituent of pI 4.7 to 4.8, accompanied by 3 acidic isoelectric variants. Upon incubation at 37 degrees C in the presence of 150 mM KCl, GFAP assembled into 10 nm-filaments.
D000818:Animals; D001369:Axons / Q000648:ultrastructure; D001923:Brain Chemistry; D002417:Cattle; D002458:Cell Fractionation; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity / Q000379:methods; D003599:Cytoskeleton / Q000648:ultrastructure; D004247:DNA / Q000031:analogs & derivatives; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins / Q000302:isolation & purification*
C033190:DNA-cellulose; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D002482:Cellulose; D004247:DNA