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, a-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. Peripherin is a class III intermediate filament subunit protein found in certain classes of neuron, most of which are located in the peripheral nervous system. Certain sensory neurons in the brain stem express peripherin normally, as do a few cortical and hippocampal neurons. Interestingly, peripherin protein is upregulated in the lower motor neurons that degenerate in Amyotrophic Lateral Sclerosis (ALS), focusing attention on peripherin as a potential contributory factor to this serious human neurodegenerative disease. Upregulation of peripherin mRNA and protein expression is seen in a variety of damage and disease states. To raise this particular antibody rat peripherin was expressed in bacteria and purified from inclusion bodies by dissolving in 6M urea followed by preparative gel electrophoresis. Thie pure protein was used to raise antibody 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. The IgY total concentration is is 26mg/ml. Suitable second antibody reagents can be obtained from many vendors including Molecular Probes and Sigma-Aldrich.

Figures:Blot in preparation. Right shows rat mixed neuron/glial cultures stained with CPCA-Peri, our chicken polyclonall antibody to peripherin (green channel) and EnCors rabbit antibody to the neurofilament subunit alpha-internexin RPCA-a-Int (green channel). These cultures contain mostly neurons which are rich in alpha-internexin, and a subgroup which have a large amount of peripherin also, such as the prominent cell in the middle of the micrograph. Since this cell expresses large amounts of peripherin and alpha-internexin, the green and red signals superimpose to produce a golden cell. 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. The IgY total concentration is is 26mg/ml. 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. Press here to download a material safety data sheet (MSDS) for sodium azide. Store at 4°C or -20°C. Avoid repeat freezing and thawing.

Suggestions for use: The IgY solution can be used at dilutions of at least 1:2,000 in immunofluorescence experiments. In western blotting using chemiluminescence it can be used at dilutions of 1:10,000 or lower. The solution has a total protein concentration of 21.5 mg/ml in phosphate buffered saline plus 10mM sodium azide.

References:

Errante LD, Wiche G and Shaw G. Distribution of plectin, an intermediate filament-associated protein, in the adult rat central nervous system J. Neurosci. Res. 37:515-528 (1994).

Price and Availability: - We currently supply 100 microliter aliquots for $200. Material is in stock and ready for immediate shipping.

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

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