The Immunogen: Neurofilaments can be defined as the intermediate or 10nm diameter filaments found in neuronal cells. They are composed a mixture of subunits which often includes the neurofilament triplet proteins, NF-L, NF-M and NF-H. Neurofilaments may also include peripherin, alpha-internexin, nestin and in some cases vimentin. Alpha-internexin is a protein running on SDS-PAGE at ~64-66 kDa and is a Class IV intermediate filament originally discovered as it copurifies with other neurofilament subunits (1). It is related to but distinct from the better known neurofilament triplet proteins, NF-L, NF-M and NF-H, having similar protein sequence motifs and a similar intron organization. It is expressed in large amounts early in neuronal development, but is downregulated in many neurons as development procedes. Many classes of mature neurons contain alpha-internexin in addition to NF-L, NF-M and NF-H. In some mature neurons alpha-internexin is the only neurofilament subunit expressed. Antibodies to alpha-internexin are therefore unique probes to study and classify neuronal types and follow their processes in sections and in tissue culture. In addition the very early developmental expression of alpha-internexin means its presence is an early and convenient diagnostic feature of neuronal progenitors cells and other cell committed to the neuronal lineage. In addition recent studies show a marked up-regulation of alpha-internexin during neuronal regeneration (2). The use of antibodies to this protein in the study of brain tumors has not been examined to date, but is likely to be of interest. The HGNC name for this protein is INA.

Left: Western blot of whole rat spinal cord homogenate stained with RPCA-a-Int, at dilution of 1:20,000. A prominent band running at ~66kDa is apparent, as well as smaller lower bands which are apparently degradation products. A minor band at ~150kDa is also seen, apparently resulting from dimerization of alpha-internexin. Right: Mixed neuron-glial cultures stained with rabbit antibody to alpha-internexin (red) and chicken antibody to peripherin CPCA-Peri (green). The alpha internexin antibody stains numerous axonal and dendritic profiles in these cultures, while peripherin antibody binds to only a subset of neurons.

Antibody Characteristics: The antibody was raised against recombinant alpha-internexin expressed in and purified from E. coli, and is similar to but not identical with antibodies described in reference 3 below. 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 1:10,000 to 20,000 for western blotting. On western blots look for a major band at 64 to 66kDa, depending on the species, the human protein being a little larger than that from rodents.

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References:

1. Pachter, J and Liem, RKH. Alpha-Internexin, a 66-kD intermediate filament-binding protein from mammalian central nervous tissues. J Cell Biol 101:1316-22 (1985)

2. McGraw et al. Axonally transported peripheral signals regulate alpha-internexin expression in regenerating motoneurons. J Neurosci 22:4955-63 (2002)

3. Evans J. et al. Characterization of mitotic neurons derived from adult rat hypothalamus and brain stem. J. Neurophysiol. 87:1076-85 2002.

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