Overview

  • Product name
    Anti-68kDa Neurofilament / NF-L antibody
    See all 68kDa Neurofilament / NF-L primary antibodies
  • Description
    Rabbit polyclonal to 68kDa Neurofilament / NF-L
  • Host species
    Rabbit
  • Specificity
    Specifically recognizes the light neurofilament subunit (~68 kDa).
  • Tested applications
    Suitable for: WB, IHC-FoFr, IHC-P, IHC-Fr, ICC/IF, IHC-FrFlmore details
  • Species reactivity
    Reacts with: Rat, Pig
    Predicted to work with: Bird, Mammals
  • Immunogen

    Full length native protein (purified) corresponding to Pig 68kDa Neurofilament/ NF-L. prepared from spinal cords by the method of Delacourte et al. and this cytoskeletal material was dissolved in 6M urea. Purified by ion exchange chromatography, then preparative gel electrophoresis.

  • General notes

    Previously labelled as 68kDa Neurofilament. 

Applications

Our Abpromise guarantee covers the use of ab9035 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB
IHC-FoFr
IHC-P
IHC-Fr
ICC/IF
IHC-FrFl
  • Application notes
    ICC/IF: 1/500.
    IHC-F: 1/1000.
    IHC-P: 1/1000.
    IHC-Fr: 1/1000.
    WB: 1/5000.

    Not yet tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • Target

    • Function
      Neurofilaments usually contain three intermediate filament proteins: L, M, and H which are involved in the maintenance of neuronal caliber.
    • Involvement in disease
      Defects in NEFL are the cause of Charcot-Marie-Tooth disease type 1F (CMT1F) [MIM:607734]. CMT1F is a form of Charcot-Marie-Tooth disease, the most common inherited disorder of the peripheral nervous system. Charcot-Marie-Tooth disease is classified in two main groups on the basis of electrophysiologic properties and histopathology: primary peripheral demyelinating neuropathy or CMT1, and primary peripheral axonal neuropathy or CMT2. Neuropathies of the CMT1 group are characterized by severely reduced nerve conduction velocities (less than 38 m/sec), segmental demyelination and remyelination with onion bulb formations on nerve biopsy, slowly progressive distal muscle atrophy and weakness, absent deep tendon reflexes, and hollow feet. CMT1F is characterized by onset in infancy or childhood (range 1 to 13 years).
      Defects in NEFL are the cause of Charcot-Marie-Tooth disease type 2E (CMT2E) [MIM:607684]. CMT2E is an autosomal dominant form of Charcot-Marie-Tooth disease type 2. Neuropathies of the CMT2 group are characterized by signs of axonal regeneration in the absence of obvious myelin alterations, normal or slightly reduced nerve conduction velocities, and progressive distal muscle weakness and atrophy.
    • Sequence similarities
      Belongs to the intermediate filament family.
    • Domain
      The extra mass and high charge density that distinguish the neurofilament proteins from all other intermediate filament proteins are due to the tailpiece extensions. This region may form a charged scaffolding structure suitable for interaction with other neuronal components or ions.
    • Post-translational
      modifications
      O-glycosylated.
      Phosphorylated in the Head and Rod regions by the PKC kinase PKN1, leading to inhibit polymerization.
    • Information by UniProt
    • Database links
    • Alternative names
      • 68 kDa neurofilament protein antibody
      • 68kDa Neurofilament antibody
      • 68kDa neurofilament protein antibody
      • CMT1F antibody
      • CMT2E antibody
      • FLJ53642 antibody
      • Light molecular weight neurofilament protein antibody
      • NEFL antibody
      • Neurofilament light antibody
      • Neurofilament light polypeptide 68kDa antibody
      • Neurofilament light polypeptide antibody
      • Neurofilament protein, light chain antibody
      • Neurofilament subunit NF L antibody
      • Neurofilament triplet L protein antibody
      • NF-L antibody
      • NF68 antibody
      • NFL antibody
      • NFL_HUMAN antibody
      see all

    Images

    • Immunohistochemistry (Free Floating) analysis of mouse cerebellum staining 68kDa Neurofilament / NF-L with ab9035 (1/5000) in red. Costained with chicken pAb to MBP (1/5000) in green and DAPI in blue. Following transcardial perfusion of mouse with 4% paraformaldehyde, brain was post fixed for 24 hours, cut to 45μM, and free-floating sections were stained with above antibodies.

    • Lane 1 : Protein ladder
      Lanes 2-5 : Anti-68kDa Neurofilament / NF-L antibody (ab9035) at 1/20000 dilution

      Lane 2 : Rat brain
      Lane 3 : Rat spinal cord
      Lane 4 : Mouse brain
      Lane 5 : Mouse spinal cord

      Observed band size: 68 kDa
      why is the actual band size different from the predicted?

    • ab9035 staining anti 68kDa Neurofilament / NF-L in mixed neuron/glia cultures from newborn rat brain by ICC/IF (Immunocytochemistry/immunofluorescence). Samples were incubated with primary antibody (1/500) (red) and co-stained with ab4573 for anti Peripherin (green).

    • ab9035 at a 1/300 dilution staining rat hippocampal organotypic slice cultures by ICC/IF. The primary antibody was incubated with the cells for 120 hours (this time allows for the antibody to penetrate a layer of glial cells that grows while the slice is in culture). Bound antibody is detected using a Cy3 conjugated Indocarbocyanine.

      See Abreview

    References

    This product has been referenced in:
    • Yadav P  et al. Neurofilament depletion improves microtubule dynamics via modulation of Stat3/stathmin signaling. Acta Neuropathol 132:93-110 (2016). WB ; Mouse . Read more (PubMed: 27021905) »
    • Clarke WT  et al. Syncoilin modulates peripherin filament networks and is necessary for large-calibre motor neurons. J Cell Sci : (2010). WB ; Mouse . Read more (PubMed: 20587592) »
    See all 3 Publications for this product

    Customer reviews and Q&As

    1-6 of 6 Abreviews or Q&A

    Application
    Immunohistochemistry (Frozen sections)
    Sample
    Chicken Tissue sections (Hindbrain section)
    Permeabilization
    Yes - PBS + 0.1% Tween20
    Specification
    Hindbrain section
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Feb 21 2014

    Answer

    Thank you for your reply.

    Trying the antibody in WB is a good alternative to find out whether there might have been a problem with it. For WB, we recommend a 1/5000 dilution, and it should recognise a band at ˜68KDa under denaturing and reducing conditions.

    I would encourage you to have a look at our online protocols to perform the assay:

    https://www.abcam.com/index.html?pageconfig=resource&rid=11375

    Please let me know how the antibody performs in this technique, and I will be more than happy to help you further.

    Read More

    Answer

    Thank you for your reply.

    I can confirm that without controls it is not possible to interpret the results. Also this antibody is not experimentallytested for mouse samples. Although it is very likely that ab9035 works with mouse samples, in this case further control would be good. For example a positive control from rat.

    Cell lines often show a peculiar expression pattern of proteins since they are not primary cells and have been cultured for a long time. Cancer cells in particular have expression pattern thatcan vary from what is expected.Therefore, as you mentioned, I suggest to use a positive control and a negative control.

    A suitable positive control would be brain or the dorsal root ganglion. Spleen could be the negative control. Please follow this link for more information on the expression pattern of this protein in rat:

    http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Rn.18568

    I hope this information is helpful and wish you good luck with your experiments.

    Read More

    Answer

    Thank you for your inquiry.

    Unfortunately, I am unable to provide a straight forward answer to your question. Since you are treating cells for neuronal differentiation and I assume that the untreated cells are supposed to be negative for the 68kDa Neurofilament, the untreated cells could be one negative control. I suggest to use a cell type as positive control that does not need treatment.

    What cells are used? Rat or pig?

    To interpret the results of any experiment, multiple controls are required:

    Positive control

    A section from a cell line or tissue known to express the protein you are detecting. A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working. It will verify that any negative results are valid.

    We recommend to check the antibody datasheet, which will often provide a suggested positive control. Always ensure the tissue or cell line you use is from a tested species. Not all the datasheets will have a suggested suitable control, and we recommend the following in these circumstances:
    Check to see if there are any Abreviews for the antibody. Any tissues, cells or lysates that have been used successfully by these customers can be considered a suitable positive control.
    Try looking at the http://expasy.org/sprot/ or http://omnigene.sourceforge.net/index.shtml database links on the datasheet. These databases will often have a list of tissues that the protein is expressed in. These can also be considered suitable positive controls.
    Check the http://www.genecards.org/ entry for the protein. This will usually provide you with relative levels of expression in various tissues.
    If you still have difficulty finding a suitable control, we recommend doing a quick literature search on http://www.ncbi.nlm.nih.gov/sites/entrez to see which tissues and cells express the protein of interest.

    Negative control (if possible)

    A section from a cell line or tissue sample known not to express the protein you are detecting. This is to check for non specific binding and false positive results.

    No primary control

    This is when the primary antibody is not added to one strip of membrane. Secondary antibody only is added. This indicates if any non specific binding or false-positivesmay be due to non specific binding ofthe secondary antibody. Antibody dilution buffer containing no antibody is used in place of the primary antibody solution at this point in the procedure. The secondary antibody is incubated on the sample in the same way as usual.

    Isotype control

    Instead of the primary antibody, aantibody of the same isotype without any specific binding capacities is used. This will serve as the background control for the specific primary antibody. We suggest to use this control when adjusting the instrument to take images of the ICC or IHC.

    If background occurs in the isotype control, the gain can be decreased until this background is gone. These setting then can be used to evaluate the staining of the specific antibody. All positive staining can now be assumed to be specific.

    I am sorry that I could not provide a straight forward answer on this occasion and hope this information is helpful.

    Read More

    Answer

    Thanks for your inquiry. I checked with the lab and this is what they had to say: "The sample was serum, but in that particular batch there was some hemolysis resulting in release of some hemoglobin from red blood cells. We try to avoid shipping material like that to you as it is a little ugly looking. We have tested the serum and the presence of the hemoglobin has absolutely no impact on the efficacy of the antibody." I hope this was helpful. Please feel free to contact me if you require further assistance.

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Rat Cell (Hippocampal Organotypic Slice Cultures)
    Specification
    Hippocampal Organotypic Slice Cultures
    Fixative
    Paraformaldehyde
    Blocking step
    Serum as blocking agent for 24 hour(s) and 0 minute(s) · Concentration: 1.5%

    Dr. Luis Craveiro

    Verified customer

    Submitted Jun 30 2006

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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