Validated using a knockout cell line

Anti-Nestin antibody [196908] (ab6320)

Knockout Tested Mouse monoclonal Nestin antibody [196908]. Validated in WB, IP, ELISA, IHC, ICC, Flow Cyt, ICC/IF and tested in Human. Cited in 25 publication(s). Independently reviewed in 5 review(s).

Reviews (5)Q&A (9)References (29)

Overview

  • Product name

    Anti-Nestin antibody [196908]
    See all Nestin primary antibodies

  • Description

    Mouse monoclonal [196908] to Nestin

  • Host species

    Mouse

  • Specificity

    Based on the result of immunocytochemistry on mouse and rat neural stem cells, this antibody has no reactivity with rodent Nestin. The clone number has been updated from (3k1) to (196908) both clone numbers name the same antibody clone.

  • Tested applications

    Suitable for: IHC-Fr, IHC - Wholemount, IHC-FoFr, ICC, Flow Cyt, ELISA, WB, IP, ICC/IFmore details

  • Species reactivity

    Reacts with: Human
    Does not react with: Mouse, Rat

  • Immunogen

    Nestin-transfected NS0 cells transfected with a human Nestin fragment aa residues 618-1618.

  • Positive control

    • Human neural progenitors and A172 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab6320 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
IHC-Fr Use a concentration of 0.1 µg/ml. When using aldehyde-based fixative, use at a concentration of 5 µg/ml (see Abreview).
IHC - Wholemount Use at an assay dependent concentration.
IHC-FoFr Use at an assay dependent concentration.
ICC Use a concentration of 5 - 10 µg/ml. Tested in human neural progenitors and A172 cells. Cells were fixed with 4% paraformaldehyde and 0.15% picric acid in PBS at RT for 20 min.
Flow Cyt Use a concentration of 25 µg/ml.

Fix and permeabilize cells.
ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
ELISA Use at an assay dependent concentration.
WB Use at an assay dependent concentration.
IP Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.

Target

  • Function

    Required for brain and eye development. Promotes the disassembly of phosphorylated vimentin intermediate filaments (IF) during mitosis and may play a role in the trafficking and distribution of IF proteins and other cellular factors to daughter cells during progenitor cell division. Required for survival, renewal and mitogen-stimulated proliferation of neural progenitor cells.

  • Tissue specificity

    CNS stem cells.

  • Sequence similarities

    Belongs to the intermediate filament family.

  • Developmental stage

    Upon terminal neural differentiation, nestin is down-regulated and replaced by neurofilaments.

  • Post-translational
    modifications

    Constitutively phosphorylated. This increases during mitosis when the cytoplasmic intermediate filament network is reorganized.
  • Information by UniProt

  • Database links

    • Alternative names

      • ESTM 46 antibody
      • FLJ 21841 antibody
      • FLJ21841 antibody
      • Intermediate filament protein antibody
      • Nbla00170 antibody
      • nes antibody
      • NEST_HUMAN antibody
      • Nestin antibody
      see all

    Images

    • Western blot - Anti-Nestin antibody [196908] (ab6320)
      Western blot - Anti-Nestin antibody [196908] (ab6320)
      All lanes : Anti-Nestin antibody [196908] (ab6320) at 1 µg

      Lane 1 : Wild-type HAP1 whole cell lysate
      Lane 2 : NES knockout HAP1 whole cell lysate

      Lysates/proteins at 20 µg per lane.


      Lanes 1 - 2: Merged signal (red and green). Green - ab6320 observed at 300 kDa. Red - loading control, ab176560, observed at 50 kDa.

      ab6320 was shown to recognize NES in wild-type HAP1 cells as signal was lost at the expected MW in NES knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and NES knockout samples were subjected to SDS-PAGE. Ab6320 and ab176560 (Rabbit anti-alpha Tubulin loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

    • Immunocytochemistry/ Immunofluorescence - Anti-Nestin antibody [196908] (ab6320)
      Immunocytochemistry/ Immunofluorescence - Anti-Nestin antibody [196908] (ab6320)

      Immunocytochemical analysis of human fetal neural progenitor cells, labeling Nestin with ab6320 (10µg/ml). Immunostaining was for 3 hours at room temperature. Counterstaining with DAPI (blue).

    • Immunohistochemistry (Frozen sections) - Anti-Nestin antibody [196908] (ab6320)
      Immunohistochemistry (Frozen sections) - Anti-Nestin antibody [196908] (ab6320)Image from Griffero F et al, J Biol Chem. 2009 Mar 13;284(11):7138-48. Epub 2009 Jan 14, Fig 1.
      Tumorigenicity of human glioma-derived cells. Representative immunohistochemical analysis of brain tumors generated after orthotropic injection of isolated human glioblastoma cells. Immunofluorescence of mouse brains cryosections labeled with anti-human nestin antibody. A, diffuse infiltration of the injected and contralateral hemisphere (GBM 1). B, cells infiltrating the striatum ipsilateral and contralateral to the side of injection (GBM 2). Scale bar, 100 µm. GBM xenografts recapitulate the morphology of corresponding tumors in human cancer patients.
      For xenograft tumor analysis, mice were sacrificed, and cryopreserved brain sections were cut using a 10-µm cryostat. Staining with hematoxylin-eosin identified sections bearing tumors. Briefly, brain sections were mounted on slides and stained with hematoxylin for 40 seconds and then counterstained with alcoholic eosin for 30 seconds. Cryosections containing tumors were permeabilized in PBS containing 0.5% Triton X-100 and
    • IHC - Wholemount - Anti-Nestin antibody [196908] (ab6320)
      IHC - Wholemount - Anti-Nestin antibody [196908] (ab6320)This image is courtesy of an anonymous Abreview
      ab6320 staining Nestin in Human glioblastoma in mouse brain by Immunohistochemistry - Wholemount.

      Samples were incubated with primary antibody (1/1000 in diluent) for 16 hours at 4°C. An HRP-conjugated goat anti-mouse polyclonal IgG was used as the secondary antibody.

    References

    This product has been referenced in:

    • Yuan Y  et al. A six-inhibitor culture medium for improving naïve-type pluripotency of porcine pluripotent stem cells. Cell Death Discov 5:104 (2019). Read more (PubMed: 31240131) »
    • Yu NH  et al. Optimal Stem Cell Transporting Conditions to Maintain Cell Viability and Characteristics. Tissue Eng Regen Med 15:639-647 (2018). Read more (PubMed: 30603585) »
    See all 29 Publications for this product

    Publishing research using ab6320? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-10 of 14 Abreviews or Q&A

    Immunohistochemistry (PFA perfusion fixed frozen sections) abreview for Anti-Nestin antibody [3k1] - Neural Stem Cell Marker

     Excellent
    Abreviews
    abreview image
    Application
    Immunohistochemistry (PFA perfusion fixed frozen sections)
    Antigen retrieval step
    None
    Sample
    Human Tissue sections (human tumor bearing mouse brain)
    Specification
    human tumor bearing mouse brain
    Blocking step
    bsa 1% + Goat serum 5% as blocking agent for 2 hour(s) and 0 minute(s) · Temperature: 20°C
    Fixative
    Paraformaldehyde
    Read More

    Mrs. Francoise Geffroy

    Verified customer

    Submitted May 02 2014

    Immunocytochemistry/ Immunofluorescence abreview for Anti-Nestin antibody [3k1] - Neural Stem Cell Marker

     Good
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Blocking step
    Serum as blocking agent for 30 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Sample
    Human Cell (hair follicle culture)
    Specification
    hair follicle culture
    Permeabilization
    Yes - methanol
    Fixative
    Paraformaldehyde
    Read More

    Mr. Heiko Locher

    Verified customer

    Submitted Sep 23 2013

    Immunocytochemistry abreview for Anti-Nestin antibody [3k1] - Neural Stem Cell Marker

     Good
    Abreviews
    This product is known to not work in this application or species.
    abreview image
    Application
    Immunocytochemistry
    Sample
    Mouse Cell (adipose derived stem cell)
    Specification
    adipose derived stem cell
    Fixative
    Formaldehyde
    Permeabilization
    No
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted May 03 2013

    Question

    One lot gives patchy staining but other lots have given perfect results for 8 months. Human neuronal precursors.

    Read More
    Answer

    Thank you very much for your call yesterday, and I apologize for the variation of results using this antibody.

    As we discussed, I'm sending two free of charge vials to Cedarlane, who will then forward the replacement vials on to you.

    Please keep me updated about the results using the new vials, and let me know if you have any questions or need anything else. Have a nice day!

    Read More

    Western blot abreview for Anti-Nestin antibody [3k1] - Neural Stem Cell Marker

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Primary glioblastoma cell lines)
    Loading amount
    25 µg
    Specification
    Primary glioblastoma cell lines
    Gel Running Conditions
    Reduced Denaturing
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: RT°C
    Read More

    Abcam user community

    Verified customer

    Submitted Aug 03 2012

    IHC - Wholemount abreview for Anti-Nestin antibody [3k1] - Neural Stem Cell Marker

     Good
    Abreviews
    abreview image
    Application
    IHC - Wholemount
    Sample
    Human Tissue (Human glioblastoma in mouse brain)
    Specification
    Human glioblastoma in mouse brain
    Read More

    Abcam user community

    Verified customer

    Submitted Jul 26 2012

    Question

    Thank you for your kind reply.
    please find attchaed protocol that you requested.
    I have tried different concentrations and incubation times but obsrved the same
    results.
    I am looking forward to hearing from you.

    Read More
    Answer

    Thank you for your reply with this information.


    Regarding your protocol - one thing that really stood out is the use of Triton as a permeabilizing agent. This detergent will also partially dissolve the nuclear membrane and will disrupt proteins when used at higher concentrations for longer periods of time. We typically recommend using it at 0.1-0.2% for 10 min only. Since both of these proteins are cytomplasmic, permeabilization with tween 20 is sufficient (I recommend 0.2 % for 10 - 15 minutes). This should decrease the amount of background and non-specific stain you are observing.


    Additionally, I would recommend optimizing the conditions with each antibody individually rather than in sequential staining.


    I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions or concerns.

    Read More

    Question


    I am using two of your products (nestin and beta III) for phenotype evaluation
    in neural stem cell culture, based on recommended dilutions and with the
    secondary antibodies listed in the attached slides. However as shown in the
    second slide (please see the attachment) both products stain whole cells (even
    nucleus) instead of staining the appropriate part. Please advise me what to do
    to not have this problem.

    Read More
    Answer

    Thank you for contacting Abcam.


    I am sorry that you have been experiencing difficulties with these antibodies in ICC. I have reviewed the images provided, but I was wondering if you would also provide a detailed summary of your protocol and sample preparation. This will help me to evaluate if there are any suggestions I can make to improve your results or if the antibodies are faulty.


    I look forward to your reply so that I may assist you further. Please do not hesitate to contact me with any additional questions or concerns.

    Read More

    Question

    Multiple bands in Western blot.

    Read More
    Answer

    Thanks for your call today and for letting us know about the problem with ab6320. As we discussed, I am sending a free of charge vial of ab22035 on the order ***, which I expect to arrive tomorrow. Please keep me updated of the results with this replacement antibody. If you have any images to send, you can just reply to this email and it will go straight to me. This will help us determine how to fix the issue with ab6320. Please let me know if you have any questions or if there is anything else that we can do for you.

    Read More

    Question

    I am currently using your ab6320 (Nestin) antibody. What are the differences between this new antibody and your ab6320? Note: I am using ab6320 on human primary cells.

    Read More
    Answer

    Thank you for your enquiry. Ab27952 is a rabbit polyclonal antibody to nestin, and ab6320 is a mouse monoclonal antibody. The immunogen used to generate ab27952 was a KLH conjugated peptide to the human nestin tail. The immunogen used to generate ab6320 were nestin-transfected NS0 cells transfected with a human Nestin fragment aa residues 618-1618. Ab27952 detects human and mouse nestin while ab6320 detects human nestin and has no reactivity with rodent nestin. Please contact us again if you have any additional questions.

    Read More

    1-10 of 14 Abreviews or Q&A

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