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).

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

  • 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.

  • 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).

  • 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
  • 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:
See all 25 Publications for this product

Customer reviews and Q&As

11-14 of 14 Abreviews or Q&A

Answer

Thank you for your patience, the originator of ab6320 is still investigating this issue. We have not received any other complaints regarding this antibody and it is a popular antibody. I can send you a replacement vial if you wish. Please let me know if you are interested.

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Answer

Thank you for the details that you have submitted and for your patience. How have you stored ab6320? We do recommend aliquoting and storing this antibody at -20°C or -80°C, and also avoiding repeated freeze / thaw cycles. Thank you, and I look forward to hearing from you.

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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.

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Answer

Thank you for your enquiry. To our knowledge, cross reactivity with pig has not yet been tested. We do not routinely offer free or trial sized samples for testing purposes. Our policy at Abcam is that if an antibody does not work as specified on the datasheet, we will offer a replacement or reimbursement. Should you decide to go ahead and purchase this product, please let us know how you get on and in return we will forward a reward of your choice, typically an Amazon gift voucher.

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11-14 of 14 Abreviews or Q&A

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