Anti-Neurofilament 200 kDa Antibody, clone N52

Neurofilaments are a type of intermediate filament that serve as major elements of the cytoskeleton supporting the axon cytoplasm. They are the most abundant fibrillar components of the axon, being on average 3-10 times more frequent than axonal microtubules. Neurofilaments (10nm in dia.) are built from three intertwined protofibrils which are themselves composed of two tetrameric protofilament complexs of monomeric proteins. The neurofilament triplet proteins (68/70, 160, and 200 kDa) occur in both the central and peripheral nervous system and are usually neuron specific. The 68/70 kDa NF-L protein can self-assemble into a filamentous structure, however the 160 kDa NF-M and 200 kDa NF-H proteins require the presence of the 68/70 kDa NF-L protein to co-assemble. Neuromas, ganglioneuromas, gangliogliomas, ganglioneuroblastomas and neuroblastomas stain positively for neurofilaments. Although typically restricted to neurons, neurofilaments have been detected in paragangliomas and adrenal and extra-adrenal pheochromocytomas. Carcinoids, neuroendocrine carcinomas of the skin, and oat cell carcinomas of the lung also express neurofilaments. For more neurofilament information see Nervous System Cell Type Specific Marker chart online under the CHEMICON Technical Support section.

MAB5266 reacts with the phophorylated and dephosphorylated H-chain of neurofilament 200kDa (NF-H) in normal tissues/extracts (Shaw, 1986). MAB5266 can be used to detect cells of neuronal origin by immunohistochemistry and Western blot. It has been reported that reactivity of MAB5266 with NF-H is blocked in cdk-5 over-expressing cells (Guidato, 1996).

Carboxy terminal tail segment of enzymatically dephosphorylated porcine H-chain.

Research Category
Neuroscience

Research Sub Category
Neurofilament & Neuron Metabolism

Neuronal & Glial Markers

This Anti-Neurofilament 200 kDa Antibody, clone N52 is validated for use in WB, IH for the detection of Neurofilament 200 kDa.

Western blot: 5-10 μg/mL

Immunohistochemistry: 5-10 μg/mL

Optimal working dilutions must be determined by end user.

Immunohistochemistry: Antibody N52 reacts with a fragment of NF-200 side-arm that is located at the end of the MPR KSP domain and which contains the consensus cdk-5 phosphorylation site, however this reactivity is abolished if the NF-200 fragment becomes phosphorylated by cdk-5 {Guidato et al, 1996}. Thus for the fullest staining and reactivity, including westerns, it is suggested that samples be treated with alkaline phosphatase prior to antibody staining. The following treatment protocol is suggested:



ProtocolPreparation of Solutions:

I. TBS: Tris-HCl, 0.1 mol/l; NaCl, 0.1 mol/l′ pH 8.0.

II. Mix alkaline phosphatase, 1 mg/ml with 1 mol/l ZnSO4,; 1mol/l MgCl2, and 1mmol/l PMSF; and dissove in TBS.

NOTE: It is necessary to dialyze the alkaline phosphatase against solution I before use.

Procedure:

Frozen sections from shock frozen tissue samples are air-dried and then fixed with acetone for 10 min at -20°C. The excess acetone is allowed to evaporate at room temperature. Incubate the sections in solutions II for 4 hours at +30oC and wash in TBS 3 times for 5 minutes each. Negative control sections are incubated in solution II with out alkaline phosphatase. Further treatment as follows:

- Cover the preparation with 10-20 μl antibody solution and incubate for 1 hr in a humid chamber.

- Immerse the slide in TBS and wash in TBS 3 times for 5 minutes each.

- Cover the preparation with 10-20 μl of a solution of anti-mouse Ig-antibody, labeled with fluorscine isothiocyanate, and incubate in a humid chamber at 37°C for 1 hour.

- Wash the slide in TBS 3 times for 5 min each.

Format: Purified

Purified immunoglobulin (Ion exchange chromatography). Liquid in 0.02M phosphate buffer, 0.25M NaCl with 0.1% sodium azide, pH 7.6

Maintain at 2-8°C in undiluted aliquots for up to 6 months. Avoid repeated freeze/thaw cycles.

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

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