The Immunogen: The spectrin family of proteins were originally discovered as major components of the submembraneous cytoskeleton of osmotically lyzed red blood cells. The lyzed blood cells could be seen as clear red blood cell shaped objects in the light microscope and were referred to as red blood cell "ghosts". The major proteins of red blood cell ghosts proved to be actin, ankyrin, band 4.1 and several other proteins, including a closely spaced pair of bands running at about 240 and 260kDa on SDS-PAGE gels. This pair of bands was named "spectrin" since they were discovered in these red blood cell ghosts (1). Later work showed that similar high molecular bands were seen in membrane preparations from other eukaryotic cell types. Work by Levine and Willard described a pair of about ~240-260 kDa molecular weight bands which were transported at the slowest rate along mammalian axons. They named these proteins "fodrin" as antibody studies showed that they were localized in the sheath under the axonal membrane, but not in the core of the axon (2; fodros is Greek for sheath). Subsequently fodrin was found to be a member of the spectrin family of proteins, and the spectrin nomenclature is now normally used (3). Spectrins form tetramers of two alpha and two beta subunits, with the alpha corresponding to the lower molecular weight ~240kDa band and the beta corresponding to the ~260kDa or in some case much larger band. Most spectrin tetramers are ~0.2m long, and each alpha and beta subunit has a cell type specific expression pattern. The basic structure of each spectrin subunit is the spectrin repeat, which is a sequence of about 110 amino acids which defines a compact domain contain three closely packed alpha-helices. Each spectrin subunit contains multiple copies of this repeat, with 20 in each of the alpha subunits. The beta I-IV subunits each contain 17 spectrin repeats, while the beta V subunit, also known as beta-heavy spectrin, contains 30 of these repeats. The various subunits also contain several other kinds of functional domain, allowing the spectrin tetramer to interact with a variety of protein, ionic and lipid targets. The alpha-subunits each contain one calmodulin like calcium binding region and one Src-homology 3 (SH3) domain, an abundant domain involved in specific protein-protein interactions. The beta subunits all have a N-terminal actin binding domain and may also have one SH3 domain and one pleckstrin homology domain, a multifunctional type of binding domain which in beta I spectrin at least binds the membrane lipid PIP2 (5). Spectrins are believed to have a function in giving mechanical strength to the plasma membrane since the tetramers associate with each other to form a dense submembraneous geodesic meshwork (3). They also bind a variety of other membrane proteins and membrane lipids, and the proteins they bind to are therefore themselves localized in the membrane. Diseases may be associated with defects in one or other of the spectrin subunits (6). For example, some forms of hereditary spherocytosis, the presence of spherical red blood cells which are prone to lysis, can be traced to mutations in some of the spectrin subunits (7). As another example, various mutations altering either the actin binding domain or repeat three of bIII spectrin are causative of one form spinocerebellar ataxia, SCA5 (8). The alpha-II subunit is widely expressed in tissues but, in the nervous system, is found predominantly in neurons. Our antibody can therefore be used to identify neurons and fragments derived from neuronal membranes in cells in tissue culture and in sectioned material. This antibody was raised against a recombinant construct containing the seventh, eight and ninth of the so-called spectrin repeats.

Left: Western blots of mouse sciatic nerve extract (lanes 1 and 2) and brain homogenate (3). A prominent band running at 240kDa respresents the intact alpha-II spectrin heavy chain. More minor bands below this likely represent in vivo proteolytic fragments of alpha-II spectrin. Right: Mixed neuron-glial cultures stained with rabbit antibody to alpha-II spectrin (green) and mouse polyclonal antibody to Nestin (red). The alpha-II spectrin antibody stains numerous axonal and dendritic profiles in these cultures, clearly revealing the submembraneous cytoskeleton. Since alpha-II spectrin is specific for neurons in the CNS, the glial cells in this culture are not recognized by this antibody.

Antibody Characteristics: The antibody was raised against a recombinant construct containing the 7th, 8th and 9th spectrin repeats of hiuman alpha-II spectrin (amino acids 676-1043). The 9th spectrin repeat also includes a Src-homology 3 domain. This construct was expressed in and purified from E. coli. The antibody is provided in the form of crude rabbit serum. Store at 4°C or -20°C. Avoid repeat freezing and thawing.

Suggestions for use: The serum can be diluted to 1:500-1,000 for immunofluorescence staining and 1:5,000-10,000 for western blotting. On western blots look for a major band at 240kDa, depending on the species.

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References:

1. Marchesi VT & Steers E Jr. Selective solubilization of a protein component of the red cell membrane. Science 159:203-4 (1968).

2. Levine J & Willard M. Fodrin: axonally transported polypeptides associated with the internal periphery of many cells. J Cell Biol. 90:631-42 (1981).

3. Bennett V & Baines AJ. Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues. Physiol Rev. 81:1353-92 (2001).

4. Djinovic-Carugo K, Gautel M, Ylänne J & Young P. The spectrin repeat: a structural platform for cytoskeletal protein assemblies. FEBS Lett. 513:119-23 (2002).

5. Wang, DS and Shaw G. The association of the C-terminal region of beta I sigma II spectrin to brain membranes is mediated by a PH domain, does not require membrane proteins, and coincides with a inositol-1,4,5 triphosphate binding site. BBRC 217:608-15 (1995).

6. Bennett V & Healy J. Organizing the fluid membrane bilayer: diseases linked to spectrin and ankyrin. Trends Mol Med 14:28-36 (2008).

7. Eber S & Lux SE. Hereditary spherocytosis--defects in proteins that connect the membrane skeleton to the lipid bilayer. Semin Hematol 41:118-41 (2004).

8. Ikeda Y. et al. Spectrin mutations cause spinocerebellar ataxia type 5. Nature Genetics 38:184-90 (2006).

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