Catalogue# CPCA-NF-H: Chicken Polyclonal Antibody to Neurofilament NF-H- NEFH

The Immunogen: Neurofilaments can be defined as the intermediate or 10nm filaments found in specifically in neuronal cells. In the electron microscope neurofilaments appears as 10nm diameter fibres of indeterminate length which generally have fine wispy protrusions from their sides. They are found particularly abundantly in axons of large projection neurons. They probably function to provide structural support for neurons and their synapses and to support the large axon diameters required for rapid conduction of impulses down axons. They are composed a mixture of subunits which usually include the three neurofilament triplet proteins, known as NF-L, NF-M and NF-H. Neurofilaments may also include smaller amounts of peripherin, alpha-internexin, nestin and in some cases vimentin. Antibodies to the various neurofilament subunits are very useful cell type markers since the proteins are among the most abundant of the nervous system, are expressed only in neurons, and are biochemically very stable. The characterization of this antibody has been published in peer-reviewed form and has been shown to be very effective in ELISA (1). The antibody has a very high titer and can be used in immunofluoresence experiments at dilutions of 1 to 1 million, and reacts primarily with the phosphorylated axonal form of NF-H. As with many antibodies to NF-H, this antibody has some cross-reactivity with phosphorylated NF-M, which has similar phosphorylation sites to NF-H. To raise this particular antibody bovine intermediate filaments were prepared from spinal cords by the glycerol polymerization method of Delacourte et al., and the cytoskeletal material was dissolved in 6M urea (2). Individual neurofilament subunits were purified by ion exchange chromatography on DEAE-cellulose and the NF-H containing fractions were concentrated and further purified by preparative gel electrophoresis on a Biorad Prepcell. The HGNC name for this protein is NEFH.

We are OEM suppliers of this antibody- in other words we manufactured it, characterized it and generated the data presented on this page. This antibody is available from several other vendors, but we can supply it more cheaply and we can provide you with more detailed information on the properties of the antibody.

Figures: CBB shows lane of Coomassie Brilliant Blue stained crude extract of rat spinal cord, with the prominent major neurofilament subunit indicated; In rodents, NF-H runs at ~200kDa, NF-M at ~145kDa and NF-L at ~68kDa; in larger species such as pig or human, NF-H runs somewhat slower at ~210kDa or 220kDa. The Chk lane shows a blot probed with chicken anti NF-H antibody CPCA-NF-H. A prominent band at an apparent SDS-PAGE molecular weight of 200kDa corresponds to phosphorylated form of NF-H. The lane marked Rab shows a similar blot probed with our rabbit antibody to NF-H, RPCA-NF-H. Right shows rat mixed neuron/glial cultures stained with mouse monoclonal antibody to neurofilament subunit NF-L MCA-7D1 (green) and CPCA-NF-H, EnCor's rabbit antibody to neurofilament NF-H. This antibody binds primarily to the phosphorylated axonal forms of NF-H, in contrast to the NF-L antibody which stains both axonal and dendritic/perikaryal neurofilaments. The NF-L antibody therefore reveals a prominent cell body in green, while the surrounding axonal profiles are orange, since the are bound by both NF-L and the chicken NF-H antibody. Blue is a DNA stain.

Antibody Characteristics: This antibody was generated in chicken by standard procedures and immunoglobulin was extracted from egg yolk. The resulting polyclonal antibody belongs to the IgY subclass. This is the chicken homologue of mammalian IgG and can be used in the same general way, with the caveat that this type of antibody does not bind either Protein A or Protein G. Suitable second antibody reagents can be obtained from many vendors including Molecular Probes and Sigma-Aldrich. Store at 4°C or -20°C. Avoid repeat freezing and thawing.

Suggestions for use: The IgY solution has an extremely high titre and can be used at dilutions of at least 1:100,000 in immunofluorescence experiments. In western blotting using chemiluminescence it can be used at dilutions of 1:1,000,000 or lower. The antibody has also been used successfully in the capture role in ELISA (1).

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1. Shaw G, Yang C, Ellis R, Anderson K, Parker Mickle J, Scheff S, Pike B, Anderson DK and Howland DR. Hyperphosphorylated neurofilament NF-H is a serum biomarker of axonal injury. Biochem Biophys Res Commun. 336:1268-1277 (2005).

2. Delacourte A, Filliatreau G, Boutteau F, Biserte G, Schrevel J. Study of the 10-nm- filament fraction isolated during the standard microtubule preparation. Biochem J. 191:543-6 (1980).

Limitations: This product is for research use only and is not approved for use in humans or in clinical diagnosis.

Price and Availability: This antibody is available for shipping now. One 50μl aliquot costs $250 US. For order form press here.

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