| Name: | Mouse Monoclonal to Neurofilament Light chain, NF-L. |
| Immunogen: | NF-L purified from pig spinal cord |
| HGNC Name: | NEFL |
| UniProt: | P07196 |
| Molecular Weight: | 68-70kDa |
| Host: | Mouse |
| Isotype: | IgG1 heavy, κ light |
| Species Cross-Reactivity: | Human, rat, mouse, cow, pig, horse |
| RRID: | AB_2737579 |
| Format: | Purified antibody at 1mg/mL in 50% PBS, 50% glycerol plus 5mM NaN3 |
| Applications: | WB, IF/ICC, IHC |
| Recommended Dilutions: | WB: 1:10,000-1:20,000. IF/ICC and IHC: 1:2,000. |
| Storage: | Store at 4°C for short term, for longer term at -20°C |
Immunostaining of a coronal section of the spinal cord of a rat given a midline C4 contusion injury three days previously. Sections were stained with RPCA-NF-L-ct in red and MCA-1B11 in green. MCA-1B11 stains prominent aggregates of material concentrated in the lateral funiculi and the dorsal columns but seen in lesser amounts throughout the section. These are degenerating and degenerated axons damaged by the C4 lesion. The RPCA-NF-L-ct antibody binds the C-terminal “tail” region of NF-L which is absent or destroyed during degeneration, so the MCA-1B11 positive profiles are largely negative for RPCA-NF-L-ct.
Western blot analysis of different tissue lysates using mouse mAb to NF-L, MCA-1B11, dilution 1:20,000 in green: [1] protein standard, [2] rat brain , [3] mouse brain and [4] cow cerebelum. Strong band at about 68-70kDa corresponds to NF-L protein, with the cow protein appearing slightly larger in molecular size as expected. Low molecular weight bands detected in cow brain sample are likely post mortem proteolytic forms of NF-L.
Mouse Monoclonal Antibody to Neurofilament NF-L
Cat# MCA-1B11
$120.00 – $800.00
Neurofilaments are the 10nm or intermediate filament proteins found specifically in neurons, and are composed predominantly of three major proteins called NF-L, NF-M and NF-H, though other filament proteins, but in certain cell types and during development α-internexin, peripherin, nestin and vimentin may be included also. The major function of neurofilaments is likely to control the diameter of large axons (1). NF-L is the neurofilament light or low molecular weight polypeptide and runs on SDS-PAGE gels at 68-70kDa with some variability across species. Antibodies to NF-L are useful for identifying neuronal cells and their processes in cell culture and sectioned material. NF-L antibody can also be useful for the visualization of neurofilament rich accumulations seen in many neurological diseases, such as Lou Gehrig’s disease (ALS), giant axon neuropathy, Charcot-Marie Tooth disease and many others (2-4). Much interest has recently been focused on the detection of NF-L released from neurons into blood and CSF as a surrogate marker of primarily axonal loss in a variety of types of CNS injury and degeneration (5, 6).
The MCA-1B11 antibody was made against a preparation of NF-L protein purified from pig spinal cord. MCA-1B11 is known to bind NF-L from a variety of species including human, rat and mouse, and the epitope is 100% conserved in all mammalian NF-L sequences, so this antibody will have wide applicability. The epitope is very similar to that of the mouse monoclonal antibody UD1 a.k.a. 47.3, the capture reagent in the NF-Light™ assay, the Quanterix Simoa™ and related NF-L assays. We recently characterized the epitopes for both antibodies used in these assays and developed our own versions of them (6, 7). Interestingly the epitopes are mostly hidden in normal neurofilaments but become accessible on degeneration, so that they are novel reagents for studies of neurodegeneration. Full details of these findings are described in our a BioRχiv and in greater detail in a peer-reviewed publication in Brain Communications. It also works well on paraffin embedded histological sections of rodent CNS tissues, including transgenic mouse models. MCA-1B11 is slightly “leaky” in that it binds normal neurofilaments when used at high concentrations but shows strong binding to degenerated material at lower antibody concentrations. Other Uman type antibodies we market are MCA-1D44 and MCA-6H63. Full details of these findings are described in a pending peer-reviewed research report and in our recent BioRχiv article. We also market several other NF-L antibodies including a rabbit and chicken polyclonal antibodies RPCA-NF-L-Degen and CPCA-NF-L-Degen and an epitope mapped mouse monoclonal MCA-DA2. Mouse select image above left for larger view.
Chromogenic Immunostaining of a formalin fixed paraffin embedded brain stem section from a transgenic mouse model of ALS stained with mouse mAb to NF-L, MCA-1B11, dilution 1:1,000, detected in DAB (brown) following the Vector Labs Mouse on Mouse (M.O.M.®) ImmPRESS® HRP method, without the antigen retrieval step. Hematoxylin (blue) was used as the counterstain. The MCA-1B11 antibody labels what are clearly degenerated axons, showing typical swollen, sinusoidal and discontinuous profiles. Note that under these conditions healthy axons are not stained. Mouse select image for larger view.
This antibody was made against NF-L purified from pig spinal cord and binds to amino acids 316-370 of human NF-L as described in our recent BioRχiv preprint. The MCA-1B11 antibody has an epitope similar to the Uman NF-Light™ assay UD1, also known as 47.3 (7). A shows diagrammatically the location of various NF-L antibody epitopes and B shows peptide sequence of the epitopes for our new antibodies. Mouse select diagram for larger view.
Western blots of Uman NF-LIGHT™ antibodies and a set of EnCor reagents on full length recombinant human PROT-r-NF-L and recombinant human NF-L amino acids 306-364 PROT-r-NF-L-Stan. Lanes labelled 1 in red are protein standards of indicated molecular weights. Lanes labelled 2 were loaded with full length recombinant human NF-L, PROT-r-NF-L, while lanes labelled 3 were loaded with PROT-r-NF-L-Stan. The full length protein runs at about 75kDa, while PROT-r-NF-L-Stan runs at about 12kDa. All five antibodies recognize both constructs. UD1 is also known as 2.1 is the detection reagent in the Uman NF-LIGHT™ assay while UD2, also known as 47.3 is the capture reagent (6). The three other lanes show results obtained with EnCor antibodies MCA-1B11, MCA-4C14 and MCA-6H63 respectively as indicated. All these antibodies binds to an epitope flanking the so-called second “stutter” in the Coil 2 region of the α-helical coiled coil “rod” region of NF-L. The binding properties are very similar to the Uman mouse monoclonal antibody UD1 also known as 2.1, as described originally in Norgren et al. 2002. This antibody is the key capture reagent in the NF-Light™ assay of Uman Diagnostics and the Quanterix Simoa™ NF-L assay.
Immunofluorescent analysis of cow cerebellum section stained with mouse mAb to NF-L, MCA-1B11, dilution 1:2,000 in green, and costained with chicken pAb to VLP1, CPCA-VLP1, dilution 1:2,000 in red. Blue is Hoechst staining of nuclear DNA. Small section of cow cerebellum was fixed in 4% paraformaldehyde for 3 days, cut to 45μM, and free-floating sections were stained with the above antibodies. At this concentration the MCA-1B11 antibody labels dendrites and axons of neuronal cells in the granular layer (lower left) and prominent basket cell axons surrounding the large Purkinje neurons. The VLP1 antibody reveals protein expressed in granule cells and in synapses of the molecular layer of the cerebellum.
1. Hoffman PN, 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. and 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).
6. Norgren N, Karlsson J, E. Rosengren L. and Stigbrand T. Monoclonal antibodies selective for low molecular weight neurofilaments. Hybrid Hybridomics 21:53-59 (2002).
7. Shaw G, et al. Uman type neurofilament light antibodies are effective reagents for the imaging of neurodegeneration. Brain Communications doi.org/10.1093/braincomms/fcad067.
