Native Pig GFAP Protein Cat# Prot-m-GFAP

      Glial Fibrillary Acidic Protein (GFAP) was discovered by Amico Bignami and coworkers as a major fibrous protein of multiple sclerosis plaques (2). It was subsequently found to be a member of the 10nm or intermediate filament protein family, specifically the intermediate filament protein family Class III, which also includes peripherin, desmin and vimentin. The one mammalian GFAP gene produces several transcripts producing several different possible protein products (1). In pigs there are two major protein products, one of 428 amino acids and a second including a 40 amino acid insert in the C-terminal region. The two proteins are of about equal abundance and run on gels at about 50kDa and 54kDa, so purified pig GFAP therefore presents as two closely spaced bands as shown in the gel image. The larger protein is in Genbank here and the lower here. Both forms bind most available GFAP antibodies including our mouse monoclonal MCA-5C10 and our rabbit and chicken polyclonals, RPCA-GFAP and CPCA-GFAP. GFAP is strongly and specifically expressed in astrocytes and certain other astroglia in the central nervous system, in satellite cells in peripheral ganglia, and in non-myelinating Schwann cells in peripheral nerves. In many damage and disease states GFAP expression is heavily upregulated in astrocytes. In addition neural stem cells frequently strongly express GFAP. Antibodies to GFAP are therefore very useful as markers of astrocytic cells and neural stem cells. In addition many types of brain tumor, presumably derived from astrocytic cells, heavily express GFAP. Alexander’s disease was recently shown to be caused by point mutations in the protein coding region of the GFAP gene (3). All forms of Alexander disease are characterized by the presence of Rosenthal fibers, which are GFAP containing cytoplasmic inclusions found in astrocytes. There has been considerable recent interest in GFAP due to potential use as a damage and degeneration biomarker, since it can be detected in blood and CSF following various kinds of CNS damage and disease states (5). This GFAP preparation is an excellent protein standard for such experiments. Since the human and rodent proteins are somewhat different in primary sequence from each other and from the pig protein, we have also generated recombinant forms of these specifically Prot-r-GFAP and Prot-r-GFAP-rat. The HGNC name for this protein is GFAP.

This protein was purified using a modification of the method of Leung and Liem (1). This involves homogenization followed by pelleting of axonal material. The axonal material is then extracted with 1% Triton X-100 to remove myelin by centrifugation several times. The pellet is then dissolved in 6M urea buffer to dissolve cytoskeletal material. This material was then mixed with hydroxyapatite which was then washed with 10mM Urea plus 10mM phosphate buffer pH=7.0. The GFAP protein does not bind to hydroxyapatite under these conditions and can be further purified using ion exchange chromatography on a DEAE column.

1. Leung, C. L. and Liem, R. K. H. Isolation of intermediate filaments. Curr. Prot. Cell Biol. 3:Unit 3.23 doi: 10.1002/0471143030.cb0323s31 (2006).
2. Bignami A, Eng LF, Dahl D, Uyeda CT. Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res. 43:429-35 1972.
3. Brenner M. et al.. Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease. Nat Genet 27:117-20 2001
4. Liem RKH, Yen SH, Salomon GD and Shelanski ML. Intermediate filaments in nervous tissues. J Cell Biol 79:637-745 (1978).
5. Moeton, M. et al. GFAP isoforms control intermediate filament network dynamics, cell morphology, and focal adhesions Cell Mol Life Sci 73:4101-4120 (2016).