EnCor Biotechnology
Recombinant Human Neurofilament NF-L (Nfl, NEFL) Fragment, Cat# PROT-r-NF-L-rct
Description
A codon optimized cDNA designed to express amino acids 256-400 of the α-helical rod region of human neurofilament NF-L, specifically the Coil 2a and Coil 2b regions, which was inserted into pET30a(+) eukaryotic expression vector, which adds an N-terminal in frame His-tag and some other vector derived sequence. This was transformed into E. coli and the recombinant protein was purified in 6M urea using immobilized metal affinity chromatography. Purified protein was diluted to 0.5mg/mL and is supplied in 6M urea. Our full length recombinant human NF-L protein PROT-r-NF-L is widely used as a protein standard in ELISA, Simoa™ and other kinds of antibody based assays for NF-L detection. This truncated product contains the epitopes for both the capture and detect NF-L monoclonal antibodies used in the Uman NF-Light Assay. The protein also contains the epitopes for our panel of degeneration specific mouse monoclonals to NF-L, MCA-6H63, MCA-1B11 and MCA-1D44. Our degeneration specific rabbit and chicken polyclonals RPCA-NF-L-Degen and CPCA-NF-L-Degen also recognize this construct, see our peer reviewed Brain Communications publication.
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| Name: | Neurofilament Light, NF-L protein fragment, Cat# Prot-r-NF-L-Rct |
| HGNC Name: | NEFL |
| UniProt: | PO7196 |
| Molecular Weight: | 22.5kDa by SDS-PAGE plus ~5kDa His Tag |
| Host: | E. coli |
| RRID: | NA |
| Format: | 0.5mg/mL in 6M urea, 10mM Phosphate pH=7.5 |
| Applications: | Protein standard for ELISA, MSD, Luminex and Simoa assays, immunogen for antibody production |
| Storage: | Stable at 4°C for several months. For longer term store at -20°C or lower |
Neurofilaments are the 10 nm or intermediate filament proteins found specifically in neurons, and are composed predominantly of four major proteins called NF-L, NF-M, NF-H and α-internexin. NF-L, NF-M and NF-H were named based on their apparent molecular weight on SDS-PAGE gels, so NF-L is low or light, NF-M is medium or middle and NF-H is high or heavy. On SDS-PAGE NF-L runs at 68-70kDa, NF-M at 145-160kDa and NF-H at 200-220kDa with some species variability, larger species tending to have larger molecules. In every case the real molecular weight is significantly lower since long acidic sequences in these molecules cause them to run aberrantly. These three proteins are major components of large diameter axons in the adult, while α-internexin is a more major component of the developing nervous system, although still present in the adult. NF-L and other neurofilament subunits accumulate in many neurological diseases, such as Lou Gehrig’s disease (ALS) and Alzheimer’s disease, and mutations in the protein coding region of the human NF-L gene cause some forms of Charcot-Marie-Tooth disease (2-4). NF-L is a very abundant protein particularly concentrated in large diameter axons and may leak into blood and CSF following various kinds of axonal injury and/or degeneration. There has therefore been much recent interest in the detection of NF-L in CSF and blood as a surrogate marker of neuronal damage and degeneration (5). NF-L is also known as NF-Light, Nfl and NEFL.
Human NF-L sequence was based on that was NP_006149.2 which was inserted into the eukaryotic expression vector pET30a(+) which adds an N terminal His-tag and some other sequence, underlined below. This sequence includes a thrombin cleavage site (blue), an S-tag affinity peptide (red) and an enterokinase cleavage site (green).
MHHHHHHSSG LVPRGSGMKE TAAAKFERQH MDSPDLGTDD DDKAMADIGS EFEFAALKDI 60
RAQYEKLAAK NMQNAEEWFK SRFTVLTESA AKNTDAVRAA KGGGAALKDI RAQYEKLAAK 120
NMQNAEEWFK SRFTVLTESA AKNTDAVRAA KGGGAALKDI RAQYEKLAAK NMQNAEEWFK 180
SRFTVLTESA AKNTDAVRAA KGGGAALKDI RAQYEKLAAK NMQNAEEWFK SRFTVLTESA 240
AKNTDAVRAA KGGGAALKDI RAQYEKLAAK NMQNAEEWFK SRFTVLTESA AKNTDAVRAA 300
K
Number of amino acids: 301
Molecular weight: 33027.11
Theoretical pI: 9.42
Amino acid composition:
Ala (A) 60 19.9%
Arg (R) 17 5.6%
Asn (N) 15 5.0%
Asp (D) 17 5.6%
Cys (C) 0 0.0%
Gln (Q) 11 3.7%
Glu (E) 24 8.0%
Gly (G) 17 5.6%
His (H) 7 2.3%
Ile (I) 6 2.0%
Leu (L) 17 5.6%
Lys (K) 33 11.0%
Met (M) 9 3.0%
Phe (F) 13 4.3%
Pro (P) 2 0.7%
Ser (S) 15 5.0%
Thr (T) 17 5.6%
Trp (W) 5 1.7%
Tyr (Y) 5 1.7%
Val (V) 11 3.7%
Total number of negatively charged residues (Asp + Glu): 41
Total number of positively charged residues (Arg + Lys): 50
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 34950
Abs 0.1% (=1 g/l) 1.058
1. Hoffman et al. Neurofilament gene expression: a major determinant of axonal caliber. PNAS 84:3472-6 (1987).
2. Perrot R, et al. Review of the Multiple Aspects of Neurofilament Functions, and their Possible Contribution to Neurodegeneration.Mol. Neurobiol. 38:27-65 (2008).
3. Lépinoux-Chambaud C. Eyer J. Review on intermediate filaments of the nervous system and their pathological alterations. Histochem. Cell Biol. 140:13-22 (2013).
4. Liu Q. et al. Neurofilamentopathy in Neurodegenerative Diseases. Open Neurol. J. 5:58–62 (2011).
5. Bacioglu M, et al. Neurofilament light chain in blood and CSF as marker of disease progression in mouse models and in neurodegenerative diseases. Neuron 91:56-66 (2016).
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