Phosphorylated Neurofilament H ELISA Kit

Analytes Available	Species Reactivity	Key Applications	Detection Methods
Phosphorylated Neurofilament (pNF-H)	B, H, M, Po, R, Fe	ELISA	Chromogenic

Analytes Available

Species Reactivity

Key Applications

Detection Methods

Phosphorylated Neurofilament (pNF-H)

B, H, M, Po, R, Fe

ELISA

Chromogenic

Description
Catalogue Number	NS170
Brand Family	Chemicon®
Trade Name	Chemicon
Description	Phosphorylated Neurofilament (pNF-H) Sandwich ELISA Kit
Background Information	The Chemicon Phosphorylated Neurofilament H (pNF-H) kit is a sandwich enzyme immunoassay (EIA), which measures pNF-H. The kit will measure pNF-H from a variety of mammalian species including human, rat, mouse, bovine, porcine, and feline. Neurofilaments are the 10nm diameter filaments which make up the major part of the cytoskeleton of neurons and are particularly concentrated in larger diameter axons. They belong to the intermediate or 10nm filament protein/gene superfamily which also includes the keratins, the major structural proteins of skin. Neurofilaments are generally regarded as being composed of three major protein subunits referred to as NF-L, NF-M and NF-H. NF-L is the "low" or "light" subunit, NF-M is the "medium" or "middle" subunit and NF-H is the "heavy" or "high" subunit. This nomenclature is based on the apparent size of these proteins on SDS-PAGE gels; NF-L is typically about 68kDa, NF-M is about 150kDa and NF-H is about 200kDa. These three proteins, referred to as the neurofilament triplet, were discovered in the 1970s as a result of studies of axonally transported proteins (Hoffman and Lasek 1975). A fourth protein, α-internexin, a.k.a NF66, was later discovered as a protein co-purifiing with the neurofilament triplet proteins (Pachter and Liem 1985). A fifth protein, peripherin, is found in some neurofilaments along with the other four proteins, mostly in the peripheral nervous system (Portier et al. 1983). Neurofilaments are major proteins of neurons and are particularly concentrated in axons. The protein NF-H has some very unusual properties. Firstly it contains 50-60 back to back hexa, hepta or octapeptide repeats each containing the sequence Lysine- Serine-Proline (KSP). The serine residues in these peptide repeats are phosporylated in vivo, and in axonal neurofilaments, these sites are heavily phosphorylated. The phosphorylated forms of NF-H are also quite resistant to proteases, suggesting that this very abundant protein might be particularly resistant to proteotytic cleavage upon release from damaged and diseased axons. This means that detection of this protein in cerebro spinal fluid (CSF) and blood would provide information about the degree of axonal injury which has occurred (reviewed in Petzold 2005). As recently shown, this protein can be detected in quite large amounts following experimental spinal cord and brain injury in rats (Shaw et al. 2005). Levels of greater than 100 ng/ml of pNF-H were detectable in blood following serious spinal cord injury and lower but still easily detectable levels were seen in blood of animals given experimental brain injury. More recent studies have revealed considerable amounts of this protein in the blood of mice transgenic for mutations of human copper/zinc superoxide dismutase 1 which are associated with amyotrophic lateral sclerosis (ALS). These mice develop an axonal degeneration pathology similar to that seen in humans with ALS, and blood pNF-H levels can be used to monitor this. Interestingly, pNF-H is detectable before the onset of obvious disease symptoms. Other studies show that pNF-H can be detected in tissue homogenates. Other experiments show that pNF-H can be detected in the plasma of humans suffering from optic neuritis (Petzold et al 2004) and in elevated levels in the cerebrospinal fluid (CSF) of individuals suffering from brain tumors and stroke (Petzold and Shaw, unpublished). Test Principle With the Chemicon pNF-H assay system, chicken polyclonal antibodies generated against pNF-H, are pre-coated onto a 96-well plate and are used to capture pNF-H from a sample. Captured pNF-H is detected using pNF-H specific rabbit polyclonal antibodies and a goat anti-rabbit alkaline phosphatase conjugate. After addition of the substrate solution the amount of pNF-H is determined. The standard curve demonstrates a direct relationship between Optical Density (OD) and pNF-H concentration: i.e., the higher the OD the higher the pNF-H concentration in the sample. Application The Chemicon pNF-H kit is designed to measure the amount of pNF-H in tissue homogenates and biological fluid (serum, plasma, and serum-free) samples from mouse and rat. There are enough reagents included in this kit for one 96-well immuno-assay plate. Running duplicate wells for samples and standards is recommended.
Materials Required but Not Delivered	1. Multi-channel or repeating pipettes 2. Pipettors & tips capable of accurately measuring 10-1000 μL 3. Graduated serological pipettes 4. 96-well microplate reader capable of measuring absorbance at 405nm 5. Graph paper for manual plotting of data 6. 1.5 mL tubes 7. Mechanical vortex 8. Two1 liter containers 9. Plate shaker (optional) 10. 3N NaOH (optional) 11. Distilled or deionized water.

Description

Catalogue Number

NS170

Brand Family

Chemicon®

Trade Name

Chemicon

Description

Phosphorylated Neurofilament (pNF-H) Sandwich ELISA Kit

Background Information

The Chemicon Phosphorylated Neurofilament H (pNF-H) kit is a sandwich enzyme immunoassay (EIA), which measures pNF-H. The kit will measure pNF-H from a variety of mammalian species including human, rat, mouse, bovine, porcine, and feline.

Neurofilaments are the 10nm diameter filaments which make up the major part of the cytoskeleton of neurons and are particularly concentrated in larger diameter axons. They belong to the intermediate or 10nm filament protein/gene superfamily which also includes the keratins, the major structural proteins of skin. Neurofilaments are generally regarded as being composed of three major protein subunits referred to as NF-L, NF-M and NF-H. NF-L is the "low" or "light" subunit, NF-M is the "medium" or "middle" subunit and NF-H is the "heavy" or "high" subunit. This nomenclature is based on the apparent size of these proteins on SDS-PAGE gels; NF-L is typically about 68kDa, NF-M is about 150kDa and NF-H is about 200kDa. These three proteins, referred to as the neurofilament triplet, were discovered in the 1970s as a result of studies of axonally transported proteins (Hoffman and Lasek 1975). A fourth protein, α-internexin, a.k.a NF66, was later discovered as a protein co-purifiing with the neurofilament triplet proteins (Pachter and Liem 1985). A fifth protein, peripherin, is found in some neurofilaments along with the other four proteins, mostly in the peripheral nervous system (Portier et al. 1983). Neurofilaments are major proteins of neurons and are particularly concentrated in axons. The protein NF-H has some very unusual properties. Firstly it contains 50-60 back to back hexa, hepta or octapeptide repeats each containing the sequence Lysine- Serine-Proline (KSP). The serine residues in these peptide repeats are phosporylated in vivo, and in axonal neurofilaments, these sites are heavily phosphorylated. The phosphorylated forms of NF-H are also quite resistant to proteases, suggesting that this very abundant protein might be particularly resistant to proteotytic cleavage upon release from damaged and diseased axons. This means that detection of this protein in cerebro spinal fluid (CSF) and blood would provide information about the degree of axonal injury which has occurred (reviewed in Petzold 2005). As recently shown, this protein can be detected in quite large amounts following experimental spinal cord and brain injury in rats (Shaw et al. 2005). Levels of greater than 100 ng/ml of pNF-H were detectable in blood following serious spinal cord injury and lower but still easily detectable levels were seen in blood of animals given experimental brain injury. More recent studies have revealed considerable amounts of this protein in the blood of mice transgenic for mutations of human copper/zinc superoxide dismutase 1 which are associated with amyotrophic lateral sclerosis (ALS). These mice develop an axonal degeneration pathology similar to that seen in humans with ALS, and blood pNF-H levels can be used to monitor this. Interestingly, pNF-H is detectable before the onset of obvious disease symptoms. Other studies show that pNF-H can be detected in tissue homogenates. Other experiments show that pNF-H can be detected in the plasma of humans suffering from optic neuritis (Petzold et al 2004) and in elevated levels in the cerebrospinal fluid (CSF) of individuals suffering from brain tumors and stroke (Petzold and Shaw, unpublished).

Test Principle With the Chemicon pNF-H assay system, chicken polyclonal antibodies generated against pNF-H, are pre-coated onto a 96-well plate and are used to capture pNF-H from a sample. Captured pNF-H is detected using pNF-H specific rabbit polyclonal antibodies and a goat anti-rabbit alkaline phosphatase conjugate. After addition of the substrate solution the amount of pNF-H is determined. The standard curve demonstrates a direct relationship between Optical Density (OD) and pNF-H concentration: i.e., the higher the OD the higher the pNF-H concentration in the sample.

Application The Chemicon pNF-H kit is designed to measure the amount of pNF-H in tissue homogenates and biological fluid (serum, plasma, and serum-free) samples from mouse and rat. There are enough reagents included in this kit for one 96-well immuno-assay plate. Running duplicate wells for samples and standards is recommended.

Materials Required but Not Delivered

1. Multi-channel or repeating pipettes
2. Pipettors & tips capable of accurately measuring 10-1000 μL
3. Graduated serological pipettes
4. 96-well microplate reader capable of measuring absorbance at 405nm
5. Graph paper for manual plotting of data
6. 1.5 mL tubes
7. Mechanical vortex
8. Two1 liter containers
9. Plate shaker (optional)
10. 3N NaOH (optional)
11. Distilled or deionized water.

Product Information
Components	Immunoplate Pre-coated with Chicken anti-pNF-H Polyclonal Antibody: (Part No. 2006263). One 96-well immunoplate pre-coated with chicken anti- pNF-H polyclonal antibody sealed in a foil pouch. Rabbit anti- pNF-H Polyclonal Antibody: (Part No. 2006919). One vial (100 μL) of rabbit anti- pNF-H polyclonal antibody. Goat anti-Rabbit, Alkaline Phosphatase Conjugated Antibody: (Part No. 2006917). One vial (10 μL) of goat anti-rabbit alkaline phosphatase conjugate. Bovine pNF-H Standard: (Part No. 2006918). One vial (5.0 ng) of bovine pNF-H standard. 20X TBS: (Part No. 2004913). One bottle (50 mL) of 20X TBS. 20% Tween-20™ : (Part No. 2004915). One vial (3 mL) of 20% Tween-20™. Elisa Assay Diluent: (Part No. 2006921) One bottle (30 mL) of Elisa assay diluent containg 0.05% Kathon as a preservative, provided in a ready to use format. pNPP Buffer: (Part No. 2006261). One bottle (12 mL) of pNPP buffer for use with 50X p-NPP, provided in a ready to use format. 50X pNPP: (Part No. 2006262). One vial (250 μL) of 50X pNPP (p-nitrophenyl phosphatase) alkaline phosphatase substrate. Plate Covers - Two Plate Covers.
Detection method	Chromogenic

Product Information

Components

Immunoplate Pre-coated with Chicken anti-pNF-H Polyclonal Antibody: (Part No. 2006263). One 96-well immunoplate pre-coated with chicken anti- pNF-H polyclonal antibody sealed in a foil pouch.
Rabbit anti- pNF-H Polyclonal Antibody: (Part No. 2006919). One vial (100 μL) of rabbit anti- pNF-H polyclonal antibody.
Goat anti-Rabbit, Alkaline Phosphatase Conjugated Antibody: (Part No. 2006917). One vial (10 μL) of goat anti-rabbit alkaline phosphatase conjugate.
Bovine pNF-H Standard: (Part No. 2006918). One vial (5.0 ng) of bovine pNF-H standard.
20X TBS: (Part No. 2004913). One bottle (50 mL) of 20X TBS.
20% Tween-20™ : (Part No. 2004915). One vial (3 mL) of 20% Tween-20™.
Elisa Assay Diluent: (Part No. 2006921) One bottle (30 mL) of Elisa assay diluent containg 0.05% Kathon as a preservative, provided in a ready to use format.
pNPP Buffer: (Part No. 2006261). One bottle (12 mL) of pNPP buffer for use with 50X p-NPP, provided in a ready to use format.
50X pNPP: (Part No. 2006262). One vial (250 μL) of 50X pNPP (p-nitrophenyl phosphatase) alkaline phosphatase substrate.
Plate Covers - Two Plate Covers.

Detection method

Chromogenic

Applications
Application	The Phosphorylated Neurofilament H (pNF-H) kit is a sandwich enzyme immunoassay (EIA), which measures pNF-H.
Key Applications	ELISA

Applications

Application

The Phosphorylated Neurofilament H (pNF-H) kit is a sandwich enzyme immunoassay (EIA), which measures pNF-H.

Key Applications

ELISA

Biological Information
Species Reactivity	Bovine Human Mouse Pig Rat Feline
Analytes Available	Phosphorylated Neurofilament (pNF-H)
Entrez Gene Number	NM_021076.2
Gene Symbol	NEFH NF-H NFH KIAA0845
Modifications	Phosphorylation
UniProt Number	P12036
UniProt Summary	FUNCTION: SwissProt: P12036 # Neurofilaments usually contain three intermediate filament proteins: L, M, and H which are involved in the maintenance of neuronal caliber. NF-H has an important function in mature axons that is not subserved by the two smaller NF proteins. SIZE: 1026 amino acids; 112480 Da PTM: There are a number of repeats of the tripeptide K-S-P, NFH is phosphorylated on a number of the serines in this motif. It is thought that phosphorylation of NFH results in the formation of interfilament cross bridges that are important in the maintenance of axonal caliber. & Phosphorylation seems to play a major role in the functioning of the larger neurofilament polypeptides (NF-M and NF-H), the levels of phosphorylation being altered developmentally and coincident with a change in the neurofilament function. DISEASE: SwissProt: P12036 # Defects in NEFH are a cause of susceptibility to amyotrophic lateral sclerosis (ALS) [MIM:105400]. ALS is a neurodegenerative disorder affecting upper and lower motor neurons, and resulting in fatal paralysis. Sensory abnormalities are absent. Death usually occurs within 2 to 5 years. The etiology is likely to be multifactorial, involving both genetic and environmental factors. SIMILARITY: SwissProt: P12036 ## Belongs to the intermediate filament family.

Biological Information

Species Reactivity

Bovine
Human
Mouse
Pig
Rat
Feline

Analytes Available

Phosphorylated Neurofilament (pNF-H)

Entrez Gene Number

NM_021076.2

Gene Symbol

NEFH
NF-H
NFH
KIAA0845

Modifications

Phosphorylation

UniProt Number

P12036

UniProt Summary

FUNCTION: SwissProt: P12036 # Neurofilaments usually contain three intermediate filament proteins: L, M, and H which are involved in the maintenance of neuronal caliber. NF-H has an important function in mature axons that is not subserved by the two smaller NF proteins.
SIZE: 1026 amino acids; 112480 Da
PTM: There are a number of repeats of the tripeptide K-S-P, NFH is phosphorylated on a number of the serines in this motif. It is thought that phosphorylation of NFH results in the formation of interfilament cross bridges that are important in the maintenance of axonal caliber. & Phosphorylation seems to play a major role in the functioning of the larger neurofilament polypeptides (NF-M and NF-H), the levels of phosphorylation being altered developmentally and coincident with a change in the neurofilament function.
DISEASE: SwissProt: P12036 # Defects in NEFH are a cause of susceptibility to amyotrophic lateral sclerosis (ALS) [MIM:105400]. ALS is a neurodegenerative disorder affecting upper and lower motor neurons, and resulting in fatal paralysis. Sensory abnormalities are absent. Death usually occurs within 2 to 5 years. The etiology is likely to be multifactorial, involving both genetic and environmental factors.
SIMILARITY: SwissProt: P12036 ## Belongs to the intermediate filament family.

Physicochemical Information
Sensitivity	Sensitivity: 0.0585 ng/mL Range of Detection: 0.0293 ng/mL to 15 ng/mL

Physicochemical Information

Sensitivity

Sensitivity: 0.0585 ng/mL
Range of Detection: 0.0293 ng/mL to 15 ng/mL

Product Usage Statements
Usage Statement	For research use only. Not for use in diagnostic procedures.

Product Usage Statements

Usage Statement

For research use only. Not for use in diagnostic procedures.

Storage and Shipping Information
Storage Conditions	Prior to use maintain the kit components at 2°-8°C and -20°C, as indicated, until expiration date. 1. Wash Buffer - After dilution maintain the Wash Solution at 2°-8°C for up to one month or until expiration date. 2. Tween-20™ - Store at room temperature upon receipt of kit. 3. Elisa Assay Buffer - Aliquot as required to avoid repeated freeze-thaw cycles. After thawing, maintain the Elisa Assay Buffer at 2°-8°C for up to one month, or until expiration date. 4. Immunoplate Pre-coated with Chicken anti- pNF-H Polyclonal Antibody - Unused strip wells may be returned to the foil pouch and stored at 2°-8°C for up to one month, or until expiration date.

Storage and Shipping Information

Storage Conditions

Prior to use maintain the kit components at 2°-8°C and -20°C, as indicated, until expiration date.

1. Wash Buffer - After dilution maintain the Wash Solution at 2°-8°C for up to one month or until expiration date.

2. Tween-20™ - Store at room temperature upon receipt of kit.

3. Elisa Assay Buffer - Aliquot as required to avoid repeated freeze-thaw cycles. After thawing, maintain the Elisa Assay Buffer at 2°-8°C for up to one month, or until expiration date.

4. Immunoplate Pre-coated with Chicken anti- pNF-H Polyclonal Antibody - Unused strip wells may be returned to the foil pouch and stored at 2°-8°C for up to one month, or until expiration date.

Packaging Information
Material Size	1 kit
Material Package	96 wells

Packaging Information

Material Size

1 kit

Material Package

96 wells

实验耗材

分子生物学试剂

蛋白与免疫学

生化试剂

细胞及检测试剂

色谱及层析

仪器设备

实验服务

办公用品

Phosphorylated Neurofilament H ELISA Kit

浏览了该商品的用户最终购买

浏览了该商品的用户还浏览了

重要规格表

我要咨询