- SMI 35 (See other available formats)
- Regulatory Status
- Other Names
- Neurofilament heavy polypeptide, NF-H, 200 kD neurofilament protein, neurofilament triplet H protein
- Mouse IgG1, κ
- Ave. Rating
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- Product Citations
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Neurofilaments (NFs) are approximately 10 nanometer intermediate filaments found in neurons. They are a major component of the neuronal cytoskeleton, and function primarily to provide structural support for the axon and to regulate the axon diameter. There are three major NF subunits, and the names given to these subunits are based upon the apparent molecular mass of the mammalian subunits on SDS-PAGE. The light or lowest neurofilament (NF-L) runs at 68-70 kD. The medium or middle (NF-M) runs at about 145-160 kD, and the heavy or highest (NF-H) runs at 200-220 kD. However, the actual molecular weight of these proteins is considerably lower due to the highly charged C-terminal regions of the molecules. The level of NF gene expression correlates with the axonal diameter, which controls how fast electrical signals travel down the axon. Mutant mice with NF abnormalities have phenotypes resembling amyotrophic lateral sclerosis. NF immunostaining is common in diagnostic neuropathology. It is useful for differentiating neurons (positive for NF) from the glia (negative for NF).Product Details
- Human, Mouse, Rat
- Antibody Type
- Host Species
- Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
- The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 488 under optimal conditions.
- 0.5 mg/ml
- Storage & Handling
- The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. Do not freeze.
IHC-P - Quality tested
SB - Reported in the literature, not verified in house
- Recommended Usage
Each lot of this antibody is quality control tested by formalin-fixed paraffin-embedded immunohistochemical staining. For immunohistochemistry, a concentration range of 5.0 - 10 µg/ml is suggested. It is recommended that the reagent be titrated for optimal performance for each application.
* Alexa Fluor® 488 has a maximum emission of 519 nm when it is excited at 488 nm.
Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.
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- Excitation Laser
Blue Laser (488 nm)
- Application Notes
Additional reported applications (for the relevant formats of this clone): include ELISA Capture1-3, immunohistochemical staining on frozen tissue sections, immunofluorescence staining, and spatial biology (IBEX)6,7.
Clone SMI 35 reacts with highly phosphorylated neurofilaments, as well as with low degrees of phosphorylation. It primarily reacts with neurofilament H and with neurofilament M to a lesser extent.
Notes: On two dimensional gels, this antibody detects a band extending from the phosphorylated neurofilament position at 200 kD (pI 5.1) toward the non-phosphorylated position at 170 kD (pI 6.2).
- Additional Product Notes
Iterative Bleaching Extended multi-pleXity (IBEX) is a fluorescent imaging technique capable of highly-multiplexed spatial analysis. The method relies on cyclical bleaching of panels of fluorescent antibodies in order to image and analyze many markers over multiple cycles of staining, imaging, and, bleaching. It is a community-developed open-access method developed by the Center for Advanced Tissue Imaging (CAT-I) in the National Institute of Allergy and Infectious Diseases (NIAID, NIH).
(PubMed link indicates BioLegend citation)
- Petzold A. 2013. J. Neuroimmunol. 262:(1-10). (ELISA)
- Lu CH, et al. 2012. PLoS One. 7:e40998. (ELISA)
- Steinacker P, et al. 2011. PLoS One. 8:e23600. (ELISA)
- Poltorak M, et al. 1993. J. Neurosci. 13:2217. (IHC-F)
- Petzold A, et al. 2011. Brain. 134:464. (WB) PubMed
- Radtke AJ, et al. 2020. Proc Natl Acad Sci U S A. 117:33455-65. (SB) PubMed
- Radtke AJ, et al. 2022. Nat Protoc. 17:378-401. (SB) PubMed
AB_2750327 (BioLegend Cat. No. 835613)
AB_2750328 (BioLegend Cat. No. 835614)
- The medium or middle NF (NF-M) runs at about 145-160 kD, and the heavy or highest NF (NF-H) runs at 200-220 kD.
Tissue Distribution: CNS, peripheral nerves and glandular cells of the prostate
Cellular Distribution: Cytoskeleton, nucleus, cytosol, and mitochondrion
- Neurofilaments are the major components of the neuronal cytoskeleton. They provide axonal support and regulate axon diameter.
- Cell bodies and dendrites are generally unstained. Other cells and tissues are unreactive except for peripheral axons.
- Cell Type
- Mature Neurons
- Biology Area
- Cell Biology, Neuroscience, Neuroscience Cell Markers
- Molecular Family
- Intermediate Filaments, Phospho-Proteins
- Antigen References
- Gene ID
- 4744 View all products for this Gene ID
- View information about Neurofilament HM NF-H NF-M Phospho on UniProt.org
- If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?
It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.
- Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?
Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.
- Are other fluorophores compatible with IBEX?
Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.
- The same antibody works in one tissue type but not another. What is happening?
Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.
- How can I be sure the staining I’m seeing in my tissue is real?
In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.
|Anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated||SMI 35||IHC-P, WB, IHC-F, ELISA|
|Purified anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated||SMI 35||IHC-P, WB, IHC-F|
|Biotin anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated||SMI 35||WB|
|Alexa Fluor® 594 anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated||SMI 35||IHC-P, ICC|
|HRP anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated||SMI 35||IHC-P, WB|
|Alexa Fluor® 647 anti NF-H/NF-M, Hypophosphorylated||SMI 35||IHC-P|
|Alexa Fluor® 488 anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated||SMI 35||IHC-P, SB|
Compare Data Across All Formats
This data display is provided for general comparisons between formats.
Your actual data may vary due to variations in samples, target cells, instruments and their settings, staining conditions, and other factors.
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Anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated
Purified anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated
Biotin anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated
Alexa Fluor® 594 anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated
HRP anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated
Alexa Fluor® 647 anti NF-H/NF-M, Hypophosphorylated
Alexa Fluor® 488 anti-Neurofilament H & M (NF-H/NF-M), Hypophosphorylated