We obtained Neuromics E18 hippocampal neurons
and grew them for seven days following the Neuromics protocol. We fixed and immunostained with CPCA-Tau using our standard immunostaining protocol
(red channel). The cells were also stained in green with MCA-BH7
, our monoclonal antibody to Ubiquitin C-terminal Hydrolase 1 (UCHL1), an abundant cytoplasmic protein of neurons which is concentrated in the perikarya. Since the perikarya contain both UCHL1 and Tau, the red and green signals superimpose, giving a yellow color, while the processes, which contain relatively much more tau, appear red. Blue stain is DAPI and reveals cell nuclei of some non neuronal cells in these cultures.
|Stripe blot of crude rat brain extract. Tau protein is expressed as up to 9 different isoforms of different molecular weight and so appears as multiple closely spaced bands covering the region of the blot from 48kDa to 67 kDa, with an additional band at 100 kDa.
||Chicken Polyclonal to Anti-Microtubule Associated Protein Tau, MAPT
||Recombinant full length version of the shortest human Tau isoform purified from E. coli.
||Antibody is supplied as an aliquot of IgY preparation.
||Human, horse, cow, pig, chicken, rat, mouse
||Western blot, IF/ICC, IHC
|Suggestions for use
||Western blot: 1:10,000. IF/ICC and IHC: 1:1,000.
||Shipped on ice. Store at 4°C. For long term storage, leave frozen at -20°C. Avoid freeze / thaw cycles.
is a relatively low molecular weight member of the microtubule associated protein
or MAP family. Most of these proteins were discovered since microtubules
can be polymerized in cell homogenates and pelleted out by centrifugation, typically taking MAP proteins with them (1,2). This early work showed that tau protein facilitated the polymerization of microtubules, and was therefore given the name τ, the Greek letter tau, since it promoted tubule formation. The protein is now usually referred to simply as tau or by the HGNC
name which is MAPT.
Tau is heavily concentrated in axons of neurons, but may also be found in dendrites and in some non neuronal cells. Much interest has focused on tau as it is a major component of the neurofibrillary tangles of Alzheimer’s disease (3,4). Tau in neurofibrillary tangles is typically heavily and aberrantly phosphorylated, and it is believed that phosphorylation may be involved in tangle formation. In addition, numerous different point mutations in the tau gene are causative of Fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17, see 5).
There is one mammalian tau gene which produces at least 9 different proteins by alternate transcription. In the central nervous system 6 isoforms predominant which either include or do not include three short exon coded inserts. These proteins range is size from 352-441 amino acids and run on SDS-PAGE gels as multiple bands ranging from 48-67 kDa. In peripheral nervous system a form called “big tau” predominates, another alternate transcript which includes a 254 amino acid insert (6). This form of tau is found in small amounts in the brain also, in cranial nerve motor nuclei and sensory processes of most sensory ganglia, and runs on SDS-PAGE with an apparent molecular weight of 100 kDa (7).
Each tau protein contains 3 or 4 copies of an 18 amino acid peptide which are responsible for binding to the microtubules and are similar to those found in MAP2 and other members of the MAP family. Tau is a highly charged acidic protein with few hydrophobic residues which belongs to the family of “intrinsically unstructured proteins“. As with GAP43, MARCKS and several other similar proteins, tau isoforms run on SDS-PAGE much more slowly than expected from their actual molecular weight.
Our antibody was made against a recombinant construct expressed in and purified from E. coli and which corresponded to the shortest version of the various tau proteins, so the antibody is expected to bind to all tau isoforms.
1. Weingarten, M. D., Lockwood, A. H., Hwo, S.-Y. and Kirschner, M. W. A protein factor essential for microtubule assembly. Proc. Nat. Acad. Sci. USA 72:1858–1862 (1975).
2. Cleveland, D. W., Hwo, S. Y., Kirschner, M. W. Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin. J Mol Biol. 116:207-25 (1977).
3. Goedert, M., Spillantini, M. G. A century of Alzheimer’s disease. Science 314:777–781 (2006).
4. Ballatore, C., Lee, V. M., Trojanowski, J. Q. Tau-mediated neurodegeneration in Alzheimer’s disease and related disorders. Nat. Rev. Neurosci. 8:663–672 (2007).
5. Wolfe, M. S. Tau mutations in neurodegenerative diseases. J. Biol. Chem. 284:6021-6025 (2009).
6. Goedert M, Spillantini MG, Crowther RA. Cloning of a big tau microtubule-associated protein characteristic of the peripheral nervous system. Proc Natl Acad Sci U S A. 89:1983-7 (1992).
7. Boyne LJ, Tessler A, Murray M, Fischer I. Distribution of Big tau in the central nervous system of the adult and developing rat. J. Comp. Neurol. 358:279-93 (1995).
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