Chicken Polyclonal Antibody to Neurofilament NF-M from EnCor Biotechnology Inc.


Catalogue CPCA-NF-M: Chicken Polyclonal Antibody to Neurofilament NF-M 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. To raise this particular antibody the C-terminal extension of rat NF-M, the so-called KE segment, was expressed in bacteria and purified from inclusion bodies by dissolving in 6M urea followed by preparative gel electrophoresis (1). 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 26mg/ml. Suitable second antibody reagents can be obtained from many vendors including Molecular Probes and Sigma-Aldrich.

Left: Western blots of homogenates of SH-SY5Y cells, a human neuroblastoma cell line. Lane A shows blotting with CPCA-NF-M, which reveals a strong NF-M band at ~150kDa. Lanes B and C are homogenates of SH-SY5Y cells which were treated with maitotoxin to activate caspase family enzymes (ref. 2). Now a ~100kDa band is seen in addition o the major NF-M band. This corresponds to the C-terminal segment of NF-M which is an in vivo calpain degradation product of human NF-M. Right: View of mixed neuron/glial cultures stained with CPCA-NF-M (red). The NF-M protein is assembled into neurofilaments which are found throughout the axons, dendrites and perikarya of these cells.
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: 1. Harris, J., Ayyub, C. and Shaw G. A molecular dissection of the carboxyterminal tails of the major neurofilament subunits NF-M and NF-H. J Neurosci Res 30:47-62 1991. 2. Shaw G, Yang C, Zhang L, Cook P, Pike B, Hill WD. Characterization of the bovine neurofilament NF-M protein and cDNA sequence, and identification of in vitro and in vivo calpain cleavage sites. Biochem Biophys Res Commun. 325:619-25 (2004). 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. ©EnCor Biotechnology Inc .