製品の概要

  • 製品名
    Anti-HEF1 antibody [2G9]
    HEF1 一次抗体 製品一覧
  • 製品の詳細
    Mouse monoclonal [2G9] to HEF1
  • 特異性
    Not tested on Sin1. This antibody mostly detects HEF1 localized at the focal adhesion sites.
  • アプリケーション
    適用あり: WB, IP, ICC/IF, IHC-Fr, IHC-P, Flow Cytmore details
  • 種交差性
    交差種: Mouse, Rat, Human
  • 免疫原

    Fusion protein, corresponding to amino acids 82-398 of Human HEF1.

  • ポジティブ・コントロール
    • 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.

製品の特性

アプリケーション

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.

アプリケーション Abreviews 特記事項
WB 1/2000 - 1/6000. Predicted molecular weight: 93 kDa.
IP 1/1000.
ICC/IF Use at an assay dependent concentration. PubMed: 19376971
IHC-Fr Use at an assay dependent concentration. PubMed: 19464348
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.

ターゲット情報

  • 機能
    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.
  • 組織特異性
    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.
  • 配列類似性
    Belongs to the CAS family.
    Contains 1 SH3 domain.
  • ドメイン
    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.
  • 翻訳後修飾
    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.
  • 細胞内局在
    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.
  • Information by UniProt
  • 参照データベース
  • 別名
    • 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

画像

  • Anti-HEF1 antibody [2G9] (ab18056) at 1/2000 dilution + whole cell lysate prepared from a murine neural stem cell line at 15 µg

    Secondary
    HRP conjugated anti mouse IgG (H+L) at 1/3000 dilution
    Developed using the ECL technique

    Predicted band size : 93 kDa
    Observed band size : 105 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 115 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 1 hour

    Image courtesy of an Abreview submitted by Melina Reisenberg.

  • 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.

  • 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.

  • 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.

  • ab18056 Immunoprecipitating Human Lung Adenocarcinoma A549 cell-line. 1000µg of cell lysate was incubated with primary antibody (1/1000) and matrix (Protein G) for 20 hours at 4°C. Treatment was 100ng/ml of HGF or EGF for 5 or 60 minutes.

    See Abreview

参考文献

This product has been referenced in:
  • Harb OA  et al. The Prognostic Role of NEDD9 and P38 Protein Expression Levels in Urinary Bladder Transitional Cell Carcinoma. J Oncol 2017:6095205 (2017). IHC-P ; Human . Read more (PubMed: 28194179) »
  • Li M  et al. High expression of HEF1 predicts a poorer prognosis of hepatocellular carcinoma: A retrospective study. Mol Clin Oncol 4:159-165 (2016). Read more (PubMed: 26893853) »

See all 14 Publications for this product

レビューと Q&A

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

Wendy Greenwood

Verified customer

投稿 Jan 16 2015

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

Prof. Reiter Lab

Verified customer

投稿 Jul 07 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (embryonic stem)
Loading amount
15 µg
Specification
embryonic stem
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Prof. Reiter Lab

Verified customer

投稿 Apr 20 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (Neural stem cell line)
Loading amount
15 µg
Specification
Neural stem cell line
Gel Running Conditions
Reduced Denaturing (7.5%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
Username

Ms. Melina Reisenberg

Verified customer

投稿 Jun 19 2009

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Rat Tissue sections (Brain)
Specification
Brain
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid pH6
Permeabilization
No
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: rt°C
Username

Mr. Carl Hobbs

Verified customer

投稿 Jan 05 2009

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Mouse brain)
Specification
Mouse brain
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citric acid pH6
Permeabilization
No
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: rt°C
Username

Mr. Carl Hobbs

Verified customer

投稿 Jan 05 2009

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Tumour: Pagets disease of the nipple)
Specification
Tumour: Pagets disease of the nipple
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated
Permeabilization
No
Blocking step
BSA as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: rt°C
Username

Mr. Carl Hobbs

Verified customer

投稿 Jan 05 2009

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 conf...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Human Cell lysate - other (Human Lung Adenocarcinoma A549 cell-line)
Total protein in input
1000 µg
Specification
Human Lung Adenocarcinoma A549 cell-line
Treatment
100ng/ml either HGF or EGF for 5 or 60 min
Immuno-precipitation step
Protein G
Username

Dr. Dean Hammond

Verified customer

投稿 Jun 07 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
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%
Username

Dr. Dean Hammond

Verified customer

投稿 May 24 2007

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

登録