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    hef1nedd-9-antibody-2g9-ab18056.pdf

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Cell Biology Apoptosis Intracellular Associated Proteins
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Anti-HEF1/NEDD-9 antibody [2G9] (ab18056)

  • Datasheet
  • SDS
Reviews (9)Q&A (1)References (25)

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Immunocytochemistry - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056)
  • Flow Cytometry - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056)

Key features and details

  • Mouse monoclonal [2G9] to HEF1/NEDD-9
  • Suitable for: ICC, IHC-P, Flow Cyt
  • Reacts with: Human
  • Isotype: IgG1

You may also be interested in

Primary
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Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
Protein
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Recombinant Human HEF1/NEDD-9 protein (ab132196)
Secondary
Product image
Goat Anti-Mouse IgG H&L (HRP) (ab205719)

View more associated products

Overview

  • Product name

    Anti-HEF1/NEDD-9 antibody [2G9]
    See all HEF1/NEDD-9 primary antibodies
  • Description

    Mouse monoclonal [2G9] to HEF1/NEDD-9
  • Host species

    Mouse
  • Specificity

    Not tested on Sin1. This antibody mostly detects HEF1 / NEDD-9 localized at the focal adhesion sites.

  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC
    Human
    IHC-P
    Human
    See all applications and species data
  • Immunogen

    Fusion protein corresponding to Human HEF1/NEDD-9 aa 82-398.

  • Positive control

    • WB: Whole cell lysate prepared from a murine neural stem cell line. ICC/IF: MCF7 cells. Flow Cytometry: A549 cells. IHC-P: Human kidney tissue.
  • General notes

    Previously labelled as HEF1

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.20
    Preservative: 0.1% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity

    Protein G purified
  • Clonality

    Monoclonal
  • Clone number

    2G9
  • Myeloma

    Sp2/0
  • Isotype

    IgG1
  • Light chain type

    kappa
  • Research areas

    • Cell Biology
    • Apoptosis
    • Intracellular
    • Associated Proteins
    • Epigenetics and Nuclear Signaling
    • Transcription
    • Other factors
    • Cancer
    • Signal transduction
    • Protein phosphorylation
    • Tyrosine kinases
    • FAK family

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 (DyLight® 488) preadsorbed (ab96879)
  • 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)
    • Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control (ab91353)
  • Recombinant Protein

    • Recombinant Human HEF1/NEDD-9 protein (ab132196)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab18056 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
Flow Cyt
Human
ICC
Human
IHC-P
Human
Application Abreviews Notes
ICC
Use at an assay dependent concentration.
IHC-P
Use at an assay dependent concentration.
Flow Cyt
Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

Notes
ICC
Use at an assay dependent concentration.
IHC-P
Use at an assay dependent concentration.
Flow Cyt
Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

Target

  • Function

    Docking protein which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. May function in transmitting growth control signals between focal adhesions at the cell periphery and the mitotic spindle in response to adhesion or growth factor signals initiating cell proliferation. May play an important role in integrin beta-1 or B cell antigen receptor (BCR) mediated signaling in B- and T-cells. Integrin beta-1 stimulation leads to recruitment of various proteins including CRK, NCK and SHPTP2 to the tyrosine phosphorylated form.
  • Tissue specificity

    Widely expressed. Higher levels detected in kidney, lung, and placenta. Also detected in T-cells, B-cells and diverse cell lines. The protein has been detected in lymphocytes, in diverse cell lines, and in lung tissues.
  • Sequence similarities

    Belongs to the CAS family.
    Contains 1 SH3 domain.
  • Domain

    Contains a central domain containing multiple potential SH2-binding sites and a C-terminal domain containing a divergent helix-loop-helix (HLH) motif. The SH2-binding sites putatively bind CRK, NCK and ABL SH2 domains. The HLH motif confers specific interaction with the HLH proteins ID2, E12 and E47. It is absolutely required for the induction of pseudohyphal growth in yeast and mediates homodimerization and heterodimerization with p130cas.
    The SH3 domain interacts with two proline-rich regions of focal adhesion kinase.
  • Post-translational
    modifications

    Cell cycle-regulated processing produces four isoforms: p115, p105, p65, and p55. Isoform p115 arises from p105 phosphorylation and appears later in the cell cycle. Isoform p55 arises from p105 as a result of cleavage at a caspase cleavage-related site and it appears specifically at mitosis. The p65 isoform is poorly detected.
    Focal adhesion kinase 1 phosphorylates the protein at the YDYVHL motif (conserved among all cas proteins). The SRC family kinases (FYN, SRC, LCK and CRK) are recruited to the phosphorylated sites and can phosphorylate other tyrosine residues. Ligation of either integrin beta-1 or B-cell antigen receptor on tonsillar B-cells and B-cell lines promotes tyrosine phosphorylation and both integrin and BCR-mediated tyrosine phosphorylation requires an intact actin network. In fibroblasts transformation with oncogene v-ABL results in an increase in tyrosine phosphorylation. Transiently phosphorylated following CD3 cross-linking and this phosphorylated form binds to CRK and C3G. A mutant lacking the SH3 domain is phosphorylated upon CD3 cross-linking but not upon integrin beta-1 cross-linking. Tyrosine phosphorylation occurs upon stimulation of the G-protein coupled C1a calcitonin receptor in rabbit. Calcitonin-stimulated tyrosine phosphorylation is mediated by calcium- and protein kinase C-dependent mechanisms and requires the integrity of the actin cytoskeleton.
  • Cellular localization

    Cytoplasm > cytoskeleton > spindle and Cytoplasm > cell cortex. Nucleus. Golgi apparatus. Cell projection > lamellipodium. Cytoplasm. Cell junction > focal adhesion. Localizes to both the cell nucleus and the cell periphery and is differently localized in fibroblasts and epithelial cells. In fibroblasts is predominantly nuclear and in some cells is present in the Golgi apparatus. In epithelial cells localized predominantly in the cell periphery with particular concentration in lamellipodia but is also found in the nucleus. Isoforms p105 and p115 are predominantly cytoplasmic and associate with focal adhesions while p55 associates with mitotic spindle.
  • Target information above from: UniProt accession Q14511 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 4739 Human
    • Omim: 602265 Human
    • SwissProt: Q14511 Human
    • Unigene: 37982 Human
    • Alternative names

      • Cas like docking antibody
      • Cas scaffolding protein family member 2 antibody
      • CAS-L antibody
      • CAS2 antibody
      • CasL antibody
      • CASL_HUMAN antibody
      • CASS2 antibody
      • Crk associated substrate related antibody
      • Crk associated substrate related protein antibody
      • CRK-associated substrate-related protein antibody
      • dJ49G10.2 (Enhancer of Filamentation 1 (HEF1)) antibody
      • dJ49G10.2 antibody
      • dJ761I2.1 (enhancer of filamentation (HEF1)) antibody
      • dJ761I2.1 antibody
      • Enhancer of filamentation 1 antibody
      • Enhancer of filamentation 1 p55 antibody
      • HEF 1 antibody
      • HEF1 antibody
      • NEDD-9 antibody
      • Nedd9 antibody
      • Neural precursor cell expressed developmentally down-regulated protein 9 antibody
      • P105 antibody
      • Renal carcinoma antigen NY-REN-12 antibody
      see all

    Images

    • Immunocytochemistry - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056)
      Immunocytochemistry - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056)

      ICC/IF image of ab18056 stained MCF7 (human breast adenocarcinoma cell line) cells. The cells were fixed in 4% formaldehyde for 10 minutes and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab18056, 1 µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1 hour. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

    • Flow Cytometry - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056)
      Flow Cytometry - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056)

      Overlay histogram showing A549 (human lung carcinoma cell line) cells stained with ab18056 (red line). The cells were fixed with 80% methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab18056, 1 µg/ 1 x 106 cells) for 30 minutes at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2 µg/ 1 x 106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in A549 cells fixed with 4% paraformaldehyde (10 minutes)/permeabilized in 0.1% PBS-Tween used under the same conditions.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HEF1/NEDD-9 antibody [2G9] (ab18056)

      IHC image of ab18056 staining in human kidney formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab18056, 10 µg/ml, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    Protocols

    • Flow cytometry protocols
    • Immunohistochemistry protocols
    • Immunocytochemistry & immunofluorescence protocols

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet
    • SDS
  • References (25)

    Publishing research using ab18056? Please let us know so that we can cite the reference in this datasheet.

    ab18056 has been referenced in 25 publications.

    • Semelakova M  et al. Vimentin and Non-Muscle Myosin IIA are Members of the Neural Precursor Cell Expressed Developmentally Down-Regulated 9 (NEDD9) Interactome in Head and Neck Squamous Cell Carcinoma Cells. Transl Oncol 12:49-61 (2019). PubMed: 30267961
    • Zhang C  et al. HEF1 regulates differentiation through the Wnt5a/ß-catenin signaling pathway in human gastric cancer. Biochem Biophys Res Commun 509:201-208 (2019). PubMed: 30579603
    • Wang Y  et al. ZMYND10, an epigenetically regulated tumor suppressor, exerts tumor-suppressive functions via miR145-5p/NEDD9 axis in breast cancer. Clin Epigenetics 11:184 (2019). PubMed: 31801619
    • Gabbasov R  et al. NEDD9 promotes oncogenic signaling, a stem/mesenchymal gene signature, and aggressive ovarian cancer growth in mice. Oncogene 37:4854-4870 (2018). PubMed: 29773902
    • Roy NH  et al. Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1. Sci Signal 11:N/A (2018). PubMed: 30538176
    View all Publications for this product

    Customer reviews and Q&As

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

    1-10 of 10 Abreviews or Q&A

    Western blot abreview for Anti-HEF1/NEDD-9 antibody [2G9]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Breast cancer cell lines MDA-MB-231, HCC1954 and M)
    Gel Running Conditions
    Reduced Denaturing (8% acrylamide gel)
    Loading amount
    20 µg
    Treatment
    TGF beta 2.5ng/ml
    Specification
    Breast cancer cell lines MDA-MB-231, HCC1954 and M
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
    Read More

    Wendy Greenwood

    Verified customer

    Submitted Jan 16 2015

    Immunocytochemistry/ Immunofluorescence abreview for Anti-HEF1 / NEDD-9 antibody [2G9]

    Inconclusive
    Abreviews
    Abreviews
    abreview image
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Mouse Cell (embryonic stem)
    Permeabilization
    Yes - 0.5% triton-x-100 in PBS
    Specification
    embryonic stem
    Blocking step
    BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 25°C
    Fixative
    Paraformaldehyde
    Read More

    Prof. Reiter Lab

    Verified customer

    Submitted Jul 07 2010

    Western blot abreview for Anti-HEF1/NEDD-9 antibody [2G9]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (embryonic stem)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    15 µg
    Specification
    embryonic stem
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Prof. Reiter Lab

    Verified customer

    Submitted Apr 20 2010

    Western blot abreview for Anti-HEF1/NEDD-9 antibody [2G9]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (Neural stem cell line)
    Gel Running Conditions
    Reduced Denaturing (7.5%)
    Loading amount
    15 µg
    Specification
    Neural stem cell line
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More

    Ms. Melina Reisenberg

    Verified customer

    Submitted Jun 19 2009

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HEF1 / NEDD-9 antibody [2G9]

    Inconclusive
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Rat Tissue sections (Brain)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citric acid pH6
    Permeabilization
    No
    Specification
    Brain
    Blocking step
    BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: rt°C
    Fixative
    Formaldehyde
    Read More

    Mr. Carl Hobbs

    Verified customer

    Submitted Jan 05 2009

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HEF1/NEDD-9 antibody [2G9]

    Inconclusive
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Mouse Tissue sections (Mouse brain)
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: Citric acid pH6
    Permeabilization
    No
    Specification
    Mouse brain
    Blocking step
    BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: rt°C
    Fixative
    Formaldehyde
    Read More

    Mr. Carl Hobbs

    Verified customer

    Submitted Jan 05 2009

    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) abreview for Anti-HEF1 / NEDD-9 antibody [2G9]

    Inconclusive
    Abreviews
    Abreviews
    abreview image
    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Sample
    Human Tissue sections (Tumour: Pagets disease of the nipple)
    Antigen retrieval step
    Heat mediated
    Permeabilization
    No
    Specification
    Tumour: Pagets disease of the nipple
    Blocking step
    BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: rt°C
    Fixative
    Formaldehyde
    Read More

    Mr. Carl Hobbs

    Verified customer

    Submitted Jan 05 2009

    Question

    It is the HEF1 antibody [2G9] (ab18056). Thanks, Jorge B. Aquino

    Read More

    Abcam community

    Verified customer

    Asked on Sep 25 2007

    Answer

    Thank you for your recent enquiry. I can confirm that the following experiments have indicated that ab18056 HEF1 antibody does not recognize p130Cas: 1) 293T cells were transfected with HA-HEF1 and HA-p130Cas. The antibody was then used to confirm that there was expression of both proteins. After stripping the blot was re-probed with 2G9 Ab and only HA-HEF1 band and endogenous HEF1 bands were observed. No bands were observed for HA-p130Cas or endogenous p130Cas. 2) MCF7 cells were treated with 3 different siRNA against HEF1, 2 different siRNA against p130Cas or scramble, or siGFP for 48h followed by lysis in M-PER buffer. Western blot analysis of endogenous protein level using 2G9 or p130Cas-directed Antibody. According to the results, [2G9] did not detect p130Cas. 3) 2G9 has been shown to recognize only HEH1 depletion upon siHEF1 treatment, but not p130Cas. Some of these results are in the following article, which I hope you will find useful: The focal adhesion scaffolding protein HEF1 regulates activation of the Aurora-A and Nek2 kinases at the centrosome. Nat Cell Biol. 2005 Oct;7(10):937-46. Epub 2005 Sep 25" I hope this information is helpful. Should you have any further questions, please do not hesitate to contact me again.

    Read More

    Abcam Scientific Support

    Answered on Sep 25 2007

    Immunoprecipitation abreview for Anti-HEF1/NEDD-9 antibody [2G9]

    Good
    Abreviews
    Abreviews
    abreview image
    Application
    Immunoprecipitation
    Sample
    Human Cell lysate - other (Human Lung Adenocarcinoma A549 cell-line)
    Total protein in input
    1000 µg
    Treatment
    100ng/ml either HGF or EGF for 5 or 60 min
    Immuno-precipitation step
    Protein G
    Specification
    Human Lung Adenocarcinoma A549 cell-line
    Read More

    Dr. Dean Hammond

    Verified customer

    Submitted Jun 07 2007

    Western blot abreview for Anti-HEF1 antibody [2G9]

    Excellent
    Abreviews
    Abreviews
    Application
    Western blot
    Sample
    Human Cell lysate - other (A549 Lung Carcinoma Cell Line)
    Loading amount
    20 µg
    Specification
    A549 Lung Carcinoma Cell Line
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
    Read More

    Dr. Dean Hammond

    Verified customer

    Submitted May 24 2007

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