Catalogue RPCA-NF-L: Polyclonal Antibody to Neurofilament Subunit NF-L-NEFL
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 include the neurofilament triplet proteins, NF-L, NF-M and NF-H. Neurofilaments may also include other intermediate filamant subunits such as α-internexin, peripherin and in developing neurons nestin and vimentin. Antibodies to neurofilament subunits are very useful cell type markers since the proteins are quite abundant, biochemically stable and found only in neurons. To make this antibody bovine intermediate filaments were prepared from spinal cords by the method of Delacourte et al. (1) and the cytoskeletal material was dissolved in 6M urea. Individual neurofilament subunits were purified by ion exchange chromatography on DEAE cellulose followed by preparative gel electrophoresis using a Biorad prepcell. The antibody recognizes purified NF-L and recognizes all mammalian and avian NF-L molecules tested to date. Mutations in the protein coding region of the human NF-L gene appear to cause some forms of Charcot-Marie-Tooth disease (2) The HGNC name for this protein is NEFL.
Left: Western blot of whole rat cerebellum homogenate stained with RPCA-NF-L in the left strip, at dilution of 1:20,000. A prominent band running with an apparent SDS-PAGE molecular weight of ~68kDa corresponds to rodent NF-L. Faint bands below this are in vivo degradation products generated by calpain digestion. The major NF-H band in human, cow and bovine run a little slower on SDS-PAGE, at about 70kDa. Right lane shows a strip incubated with a new antibody against neuron specific enolase, which reveals a major band at 47kDa. Right: Mixed neuron/glia cultures from newborn rat brain stained with MCA-7C5 antibody to peripherin (green) and rabbit polyclonal antibody to NF-L RPCA-NF-L (red channel). A class of large neurons, like the one in the middle of this image, contain peripherin, while the majority of neurons and their processes contain NF-L and not peripherin. Interestingly, the periperin positive cells often contain a cytoplasmic inclusion next to the nucleus which stains for both peripherin and NF-L, and so appears golden in this kind of image. The blue channel reveals the localization of DNA.
Antibody Characteristics: To raise this antibody pig intermediate filaments were prepared from spinal cords by the method of Delacourte et al. (2) and this cytoskeletal material was dissolved in 6M urea. NF-L was purified by ion exchange chromatography, followed by preparative gel electrophoresis. Store at 4°C or -20°C. Avoid repeat freezing and thawing.
Suggestions for use: Try at dilutions of 1:500 to 1:1,000 for immunofluorescence, and 1:5,000 for ABC or other enzyme linked immunocytochemical procedures. For western blots try at 1:20,000.
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References:
1. Delacourte A, Filliatreau G, Boutteau F, Biserte G and Schrevel J. Study of the 10-nm-filament fraction isolated during the standard microtubule preparation. Biochem J. 191:543-6. (1980).
2. Mersiyanova IV, Perepelov AV, Polyakov AV, Sitnikov VF, Dadali EL, Oparin RB, Petrin AN and Evgrafov OV. A new variant of Charcot-Marie-Tooth disease type 2 is probably the result of a mutation in the neurofilament-light gene. Am. J. Hum. Genet. 67:37-46, (2000).
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Limitations: This product is for research use only and is not approved for use in humans or in clinical diagnosis.
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