Recombinant Human NF-L (Nfl, NEFL) Assay Standard Cat# PROT-r-NF-L-Stan

      Neurofilaments are the 10nm 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. 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 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).

      Human NF-L sequence was based on that was NP_006149.2 which was inserted into the eukaryotic expression vector pET29a(+) which adds an C terminal His-tag and some other sequence, shown in red below. The sequence in black is amino acids 306-364, the region of human NF-L including the epitopes for both monoclonal antibodies employed in the Uman NF-LIGHT™ and Simoa™ assays and also our novel antibodies raised against this region. The sequence also includes two tryptophan residues, shown in blue and underllned, which were added to ensure accurate spectrophotometric quantification at a wavelength of 280nm.

MKETAAAKFE RQHMDSPDLG TLVPRGSMAD IGSEFWSESR RLLKAKTLEI EACRGMNEAL 60
EKQLQELEDK QNADISAMQD TINKLENELR TTKSEWVDKL AAALEHHHHH H

Number of amino acids: 111
Molecular weight: 12705.26
Theoretical pI: 5.60

Amino acid composition:
Ala (A) 12 10.8%
Arg (R) 6 5.4%
Asn (N) 4 3.6%
Asp (D) 7 6.3%
Cys (C) 1 0.9%
Gln (Q) 5 4.5%
Glu (E) 14 12.6%
Gly (G) 4 3.6%
His (H) 7 6.3%
Ile (I) 4 3.6%
Leu (L) 12 10.8%
Lys (K) 9 8.1%
Met (M) 5 4.5%
Phe (F) 2 1.8%
Pro (P) 2 1.8%
Ser (S) 7 6.3%
Thr (T) 6 5.4%
Trp (W) 2 1.8%
Tyr (Y) 0 0.0%
Val (V) 2 1.8%


Total number of negatively charged residues (Asp + Glu): 21
Total number of positively charged residues (Arg + Lys): 15

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 11000
Abs 0.1% (=1 g/l) 0.866, assuming all pairs of Cys residues form cystines

Ext. coefficient 11000
Abs 0.1% (=1 g/l) 0.866, assuming all Cys residues are reduced