Product nameAnti-HEF1/NEDD-9 antibody [2G9]
See all HEF1/NEDD-9 primary antibodies
DescriptionMouse monoclonal [2G9] to HEF1/NEDD-9
Not tested on Sin1. This antibody mostly detects HEF1 / NEDD-9 localized at the focal adhesion sites.
Tested applicationsSuitable for: WB, IP, ICC/IF, IHC-Fr, IHC-P, Flow Cytmore details
Species reactivityReacts with: Mouse, Rat, Human
Fusion protein corresponding to Human HEF1/NEDD-9 aa 82-398.
- 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.
Previously labelled as HEF1
Storage instructionsShipped 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 bufferpH: 7.20
Preservative: 0.1% Sodium azide
Concentration information loading...
PurityProtein G purified
Light chain typekappa
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.
|WB||1/2000 - 1/6000. Predicted molecular weight: 93 kDa.|
|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.
FunctionDocking 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 specificityWidely 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 similaritiesBelongs to the CAS family.
Contains 1 SH3 domain.
DomainContains 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.
modificationsCell 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 localizationCytoplasm > 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
- Cas like docking antibody
- Cas scaffolding protein family member 2 antibody
- CAS-L antibody
Anti-HEF1/NEDD-9 antibody [2G9] (ab18056) at 1/2000 dilution + whole cell lysate prepared from a murine neural stem cell line at 15 µg
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
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.
This product has been referenced in:
- 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). Read more (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). Read more (PubMed: 30579603) »