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    nestin-antibody-196908-ab6320.pdf

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Neuroscience Cell Adhesion Proteins Cytoskeletal Proteins Intermediate Filaments
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Validated using a knockout cell line

Anti-Nestin antibody [196908] (ab6320)

  • Datasheet
  • SDS
Reviews (5)Q&A (3)References (35)

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

Key features and details

  • Mouse monoclonal [196908] to Nestin
  • Suitable for: WB, ICC/IF
  • Knockout validated
  • Reacts with: Human
  • Isotype: IgG1

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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 & Species

    Application Species
    ICC/IF
    Human
    WB
    Human
    See all applications and species data
  • Immunogen

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

  • Positive control

    • Human neural progenitors and A172 cells.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer

    pH: 7.40
    Constituents: PBS, 5% Trehalose
  • Concentration information loading...
  • Purity

    Protein G purified
  • Clonality

    Monoclonal
  • Clone number

    196908
  • Myeloma

    NS0
  • Isotype

    IgG1
  • Research areas

    • Neuroscience
    • Cell Adhesion Proteins
    • Cytoskeletal Proteins
    • Intermediate Filaments
    • Neuroscience
    • Cell Type Marker
    • Neural Stem Cell marker
    • Cardiovascular
    • Angiogenesis
    • Angiogenic Factors
    • Neuroscience
    • Neurology process
    • Neurogenesis
    • Stem Cells
    • Lineage Markers
    • Ectoderm
    • Stem Cells
    • Neural Stem Cells
    • Intracellular
    • Cancer
    • Tumor biomarkers
    • Tumor antigens
    • Stem Cells
    • Neural Stem Cells
    • Neuron Restricted Lineage
    • Cancer
    • Invasion/microenvironment
    • Angiogenesis
    • Other
    • Developmental Biology
    • Lineage specification
    • Ectoderm

Associated products

  • Compatible Secondaries

    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) (ab150113)
    • Goat Anti-Mouse IgG H&L (HRP) (ab205719)
    • Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Mouse IgG1, kappa monoclonal [15-6E10A7] - Isotype Control (ab170190)

Applications

The Abpromise guarantee

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.

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
ICC/IF
Human
WB
Human
Application Abreviews Notes
WB (1)
Use at an assay dependent concentration. Predicted molecular weight: 177 kDa.
ICC/IF (1)
Use at an assay dependent concentration.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 177 kDa.
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.
  • Target information above from: UniProt accession P48681 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 10763 Human
    • Omim: 600915 Human
    • SwissProt: P48681 Human
    • Unigene: 527971 Human
    • 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.

      Predicted band size: 177 kDa



      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).

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet
    • SDS
  • References (35)

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

    ab6320 has been referenced in 35 publications.

    • Zhang J  et al. Biological Characterization and Therapeutics for Subscalp Recurrent in Intracranial Glioblastoma. Onco Targets Ther 13:9085-9099 (2020). PubMed: 32982297
    • Jiang Y  et al. Overexpression of Limb-Bud and Heart (LBH) promotes angiogenesis in human glioma via VEGFA-mediated ERK signalling under hypoxia. EBioMedicine 48:36-48 (2019). PubMed: 31631037
    • Wen N  et al. Bromodomain inhibitor jq1 induces cell cycle arrest and apoptosis of glioma stem cells through the VEGF/PI3K/AKT signaling pathway. Int J Oncol 55:879-895 (2019). PubMed: 31485609
    • Jiang Y  et al. NFAT1-Mediated Regulation of NDEL1 Promotes Growth and Invasion of Glioma Stem-like Cells. Cancer Res 79:2593-2603 (2019). PubMed: 30940662
    • 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). PubMed: 31240131
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-8 of 8 Abreviews or Q&A

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

    Excellent
    Abreviews
    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
    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
    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

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

    Good
    Abreviews
    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
    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

    Abcam community

    Verified customer

    Asked on Mar 15 2012

    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

    Abcam Scientific Support

    Answered on Mar 15 2012

    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

    Abcam community

    Verified customer

    Asked on Jan 13 2006

    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

    Abcam Scientific Support

    Answered on Jan 13 2006

    Question

    Not sure if this is the right place to send this email but I am having problems with the Nestin antibody for FACS analysis (ab6320).I was womdering if you could tell me your correct protocol for using this antibody to stain cells for FACS analysis e.g. such as fixation and permebilisation method. Thank you for your time,

    Read More

    Abcam community

    Verified customer

    Asked on Nov 12 2004

    Answer

    Thank you for your enquiry and your interest in our products. There is no specific protocol for this anti-Nestin monoclonal antibody. Whatever cell line is being studied, the cells must be permabilized and fixed for FACS. Here we send our general suggested protocol for Flow Cytometry: Materials and Equipment Sample cells Vortex mixer 12x75mm test tubes Flow cytometer PBS/0.1% sodium azide/ 1% FBS PBS, 0.5% Paraformaldehyde, 4° C Fluorescent-labeled Antibody Pipettes Centrifuge Ice PBS/ sodium azide at 4o C Preparation of Cells 1. Prepare the desired biological cells according to the appropriate protocol. Adjust the concentration of the cells to 2x10e7 cells per ml (or 1x10e6 cells per 50ul: each test consists of 1x10e6 cells), diluting with PBS/0.1% sodium azide / 1% FBS or BSA, as necessary. The cells can be isolated up to 4 hours before being stained as long as they are kept on ice. Note: In some cases, use of a non-specific binding or blocking agent may be desired. If so, add the blocking agent and incubate at RT for 10 minutes prior to the addition of a antibody to the cells 2. Dilute the fluorophore conjugated antibody appropriately according to the specific product recommendation for the staining being done. Use PBS/0.1% sodium azide/ 1% FBS for the dilution. Typically, 0.125-0.5ul of conjugated monoclonal antibody stains 10e6 cells. Dilute the antibody so that a volume of 10-20ul are added to the cells. 3. Pipette 50ul of the previously isolated biological cells into the bottom of each labeled 12x75mm test tube. Add the appropriate volume of diluted antibody and mix gently. Incubate the cells with the antibody at 2-8° C for 30 minutes in the dark. All conjugated antibodies ( FITC, R-PE, etc.) for double or triple staining can be added simultaneously at this point and do not require additional incubations. 4. After incubation remove the unbound antibody from the cells by washing with 1ml of PBS/0.1% sodium azide/ 1% FBS. Pipette the PBS/0.1% sodium azide/ 1% FBS into each tube, vortex, then centrifuge at 350xg for 5-7 minutes at 2-8°C. Carefully aspirate the supernatant leaving about 50-100ul in the bottom of each tube. Repeat this process for a total of 3 washes. 5. If flow cytometry is to be performed the same day, resuspend the cells in 0.5ml of cold PBS/0.1% sodium azide/ 1% FBS after aspirating the supernatant. Gently vortex and analyze the cells. If analysis will not be performed the same day, the cells may be fixed in 0.5ml cold PBS with 0.5% paraformaldehyde and store at 2-8 °C in the dark in buffer containing 0.1% sodium azide for as long as 48 hours. Indirect Staining of Extracellular Proteins If an unconjugated antibody is being used with double or triple staining then the primary antibody should be incubated for 30 minutes at 4 °C and removed by washing with PBS/0.1% sodium azide/ 1% FBS. At the time, add any conjugated antibodies and repeat staining protocol. In general, direct staining is overwhelmingly preferable. Flow Cytometry for Intracellular Antigens Similarly, a collection of cell types can be sorted of the basis of specific intracellular antigen using Flow Cytometric methods. Cell Fixation and Permeabilization 1. Wash the cells twice with cold PBS. Temperature control at this point is of vital importance. Note: Cells should be kept on ice anytime they are not spinning and centrifugation in a thermostated centrifuge at 4 °C is greatly preferred. 2. Fix the cells with 1% paraformaldehyde in cold PBS for 15 minutes (4 °C). It is very important to assure that the cells are uniformly resuspended during fixation. 3. Wash the cells twice with cold PBS. 4. Permeabilize the cells with 100% ice-cold methanol added dropwise while the cells are gently vortexing. Again, it is vitally important that the cells are uniformly suspended. Allow the soak to soak in cold methanol for 15 minutes. 5. Wash the cells twice with cold PBS. Antibody Incubation 1. Add approximately 1 x 106 cells to each flow cytometry tube and wash with 1 ml of 0.1% saponin or Tween 20 diluted in PBS with 2% FBS added. 2. Resuspend cells in 1 ml 0.1% saponin(Tween 20) + 2% FBSand allow to incubate for 30 minutes blocking at room temperature. This step is to block non-specific binding. 3. Add 20 ul of antibody diluted to the recommended concentration in 100 ul of the above blocking solution and incubate for 20 minutes at room temperature. 4. If the primary antibody is not conjugated with a fluorochrome, then a second incubation with a fluorochrome-conjugated secondary antibody will be necessary. Incubate 20 minutes in blocking solution at room temperature. 5. Wash the cells once with blocking buffer, then finally with PBS. 6. Resuspend the cells in 500 ul PBS and run on flow cytometer. We hope this information will be useful for you.

    Read More

    Abcam Scientific Support

    Answered on Nov 17 2004

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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