Human/Mouse Phospho-HGFR/c-MET (Y1234/Y1235) Antibody

AF2480 7 Citations Datasheet / COA / SDS

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Detection of Human Phospho-HGF R/c-MET (Y1234/Y1235) by Western Blot.

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Citations (7)

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Summary Data Examples Preparation and Storage Reconstitution Calculator Background Product Datasheets Related Research Areas

Human/Mouse Phospho-HGFR/c-MET (Y1234/Y1235) Antibody Summary

Species Reactivity

Human, Mouse

Specificity

Detects human and mouse HGF R/c-MET when phosphorylated at Y1234/Y1235 in Western blots.

Source

Polyclonal Rabbit IgG

Purification

Antigen Affinity-purified

Immunogen

Phosphopeptide containing human HGF R/c-MET Y1234/1235 sites

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied as a 0.2 µm filtered solution in PBS.

Label

Unconjugated

Applications

Recommended Concentration

Sample

Western Blot

0.5 µg/mL

See below

Simple Western

5 µg/mL

See below

Immunohistochemistry

5-15 µg/mL

See below

CyTOF-reported

Brodie, T.M. et al. (2018) Cytometry PartA. 93: 406. Ready to be labeled using establishedconjugation methods. No BSA or other carrier proteins that could interfere withconjugation.

Immunocytochemistry

5-15 µg/mL

See below

Intracellular Staining by Flow Cytometry

2.5 µg/10⁶ cells

See below

Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.

Data Examples

Western Blot

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Detection of Human Phospho-HGF R/c-MET (Y1234/Y1235) by Western Blot. Western blot shows Goat Anti-Human HGF R/c-MET Antigen Affinity-purified Polyclonal Antibody (Catalog # AF276) immunoprecipitate of MDA‑MB‑468 human breast cancer cell line untreated (-) or treated (+) with 100 μM pervanadate (PV) for 10 minutes. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human/Mouse Phospho-HGF R/c‑MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band was detected for Phospho-HGF R/c‑MET (Y1234/Y1235) at approximately 145 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Immunocytochemistry

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HGF R/c‑MET in SH-4 Human Cell Line. HGF R/c‑MET was detected in immersion fixed SH-4 human melanoma cell line stimulated with HGF (left panel; positive staining) and non-stimulated (right panel; negative staining) using Rabbit Anti-Human/Mouse Phospho-HGF R/c‑MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480) at 15 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rabbit IgG Secondary Antibody (red; Catalog # NL004) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Immunohistochemistry

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HGF R/c-MET in Mouse Embryo. HGF R/c-MET was detected in immersion fixed frozen sections of mouse embryo (13 d.p.c.) using Rabbit Anti-Human/Mouse Phospho-HGF R/c-MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Rabbit HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS005) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Immunohistochemistry

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HGF R/c‑MET in Human Renal Cell Carcinoma Tissue. HGF R/c‑MET was detected in immersion fixed paraffin-embedded sections of human renal cell carcinoma tissue using Rabbit Anti-Human/Mouse Phospho-HGF R/c‑MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480) at 15 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Rabbit IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC003). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Intracellular Staining by Flow Cytometry

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Detection of HGF R/c‑MET in pervanadate-treated MCF‑7 Human Cell Line by Flow Cytometry. MCF‑7 human breast cancer cell line was unstimulated (light orange open histogram) or treated with 100 μM pervanadate for 10 minutes (dark orange filled histogram), then stained with Rabbit Anti-Human/Mouse Phospho-HGF R/c‑MET (Y1234/Y1235) Antigen Affinity‑purified Polyclonal Antibody (Catalog # AF2480), or control antibody (Catalog # AB‑105‑C, blue open histogram), followed by Phycoerythrin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0110). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with methanol.

Simple Western

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Detection of Human Phospho-HGF R/c‑MET (Y1234/Y1235) by Simple Western^TM. Simple Western lane view shows lysates of MDA‑MB‑468 human breast cancer cell line untreated (-) or treated (+) with 100 µM Pervanadate (PV) for 10 minutes, loaded at 0.2 mg/mL. A specific band was detected for HGF R/c‑MET at approximately 156 kDa (as indicated) using 5 µg/mL of Rabbit Anti-Human/Mouse Phospho-HGF R/c‑MET (Y1234/Y1235) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2480). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Reconstitution Calculator

Preparation and Storage

Reconstitution

Reconstitute at 0.2 mg/mL in sterile PBS.

Reconstitution Buffer Available

Reconstitution Buffer 1 (PBS)

Shipping

The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.

12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.

Background: HGFR/c-MET

HGF R, also known as Met (from N-methyl-N’-nitro-N-nitrosoguanidine induced), is a glycosylated receptor tyrosine kinase that plays a central role in epithelial morphogenesis and cancer development. HGF R is synthesized as a single chain precursor which undergoes cotranslational proteolytic cleavage. This generates a mature HGF R that is a disulfide-linked dimer composed of a 50 kDa extracellular alpha chain and a 145 kDa transmembrane beta chain (1, 2). The extracellular domain (ECD) contains a seven bladed beta -propeller sema domain, a cysteine-rich PSI/MRS, and four Ig-like E-set domains, while the cytoplasmic region includes the tyrosine kinase domain (3, 4). Proteolysis and alternate splicing generate additional forms of human HGF R which either lack of the kinase domain, consist of secreted extracellular domains, or are deficient in proteolytic separation of the alpha and beta chains (5‑7). The sema domain, which is formed by both the alpha and beta chains of HGF R, mediates both ligand binding and receptor dimerization (3, 8). Ligand-induced tyrosine phosphorylation in the cytoplasmic region activates the kinase domain and provides docking sites for multiple SH2-containing molecules (9, 10). HGF stimulation induces HGF R downregulation via internalization and proteasome-dependent degradation (11). In the absence of ligand, HGF R forms noncovalent complexes with a variety of membrane proteins including CD44v6, CD151, EGF R, Fas, Integrin alpha 6/ beta 4, Plexins B1, 2, 3, and MSP R/Ron (12‑19). Ligation of one complex component triggers activation of the other, followed by cooperative signaling effects (12‑19). Formation of some of these heteromeric complexes is a requirement for epithelial cell morphogenesis and tumor cell invasion (12, 16, 17). Paracrine induction of epithelial cell scattering and branching tubulogenesis results from the stimulation of HGF R on undifferentiated epithelium by HGF released from neighboring mesenchymal cells (20). Genetic polymorphisms, chromosomal translocation, overexpression, and additional splicing and proteolytic cleavage of HGF R have been described in a wide range of cancers (1). Within the ECD, human HGF R shares 86%‑88% aa sequence identity with canine, mouse, and rat HGF R.

References

Birchmeier, C. et al. (2003) Nat. Rev. Mol. Cell Biol. 4:915.
Corso, S. et al. (2005) Trends Mol. Med. 11:284.
Gherardi, E. et al. (2003) Proc. Natl. Acad. Sci. 100:12039.
Park, M. et al. (1987) Proc. Natl. Acad. Sci. 84:6379.
Crepaldi, T. et al. (1994) J. Biol. Chem. 269:1750.
Prat, M. et al. (1991) Mol. Cell. Biol. 12:5954.
Rodrigues, G.A. et al. (1991) Mol. Cell. Biol. 11:2962.
Kong-Beltran, M. et al. (2004) Cancer Cell 6:75.
Naldini, L. et al. (1991) Mol. Cell. Biol. 11:1793.
Ponzetto, C. et al. (1994) Cell 77:261.
Jeffers, M. et al. (1997) Mol. Cell. Biol. 17:799.
Orian-Rousseau, V. et al. (2002) Genes Dev. 16:3074.
Klosek, S.K. et al. (2005) Biochem. Biophys. Res. Commun. 336:408.
Jo, M. et al. (2000) J. Biol. Chem. 275:8806.
Wang, X. et al. (2002) Mol. Cell 9:411.
Trusolino, L. et al. (2001) Cell 107:643.
Giordano, S. et al. (2002) Nat. Cell Biol. 4:720.
Conrotto, P. et al. (2004) Oncogene 23:5131.
Follenzi, A. et al. (2000) Oncogene 19:3041.
Sonnenberg, E. et al. (1993) J. Cell Biol. 123:223.

Long Name

Hepatocyte Growth Factor Receptor

Entrez Gene IDs

4233 (Human); 17295 (Mouse)

Alternate Names

AUTS9; cMET; c-MET; EC 2.7.10; EC 2.7.10.1; hepatocyte growth factor receptor; HGF R; HGF receptor; HGF/SF receptor; HGFR; Met (c-Met); met proto-oncogene (hepatocyte growth factor receptor); met proto-oncogene tyrosine kinase; MET; oncogene MET; Proto-oncogene c-Met; RCCP2; Scatter factor receptor; SF receptor; Tyrosine-protein kinase Met

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Citations for Human/Mouse Phospho-HGFR/c-MET (Y1234/Y1235) Antibody

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

7 Citations: Showing 1 - 7
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Reviving oncogenic addiction to MET bypassed by BRAF (G469A) mutation
Authors: AR Virzì, A Gentile, S Benvenuti, PM Comoglio
Proc. Natl. Acad. Sci. U.S.A., 2018;0(0):.
Species: Mouse
Sample Types: Whole Tissue
Applications: IHC
Pulmonary pericytes regulate lung morphogenesis
Authors: K Kato, R Diéguez-Hu, DY Park, SP Hong, S Kato-Azuma, S Adams, M Stehling, B Trappmann, JL Wrana, GY Koh, RH Adams
Nat Commun, 2018;9(1):2448.
Species: Mouse
Sample Types: Cell Lysates
Applications: Western Blot
Dual MET/EGFR therapy leads to complete response and resistance prevention in a MET-amplified gastroesophageal xenopatient cohort
Oncogene, 2016;0(0):.
Species: Human
Sample Types: Whole Tissue
Applications: IHC-P
Targeting MET kinase with the small-molecule inhibitor amuvatinib induces cytotoxicity in primary myeloma cells and cell lines.
Authors: Phillip C, Zaman S, Shentu S, Balakrishnan K, Zhang J, Baladandayuthapani V, Taverna P, Redkar S, Wang M, Stellrecht C, Gandhi V
J Hematol Oncol, 2013;6(0):92.
Species: Human
Sample Types: Whole Cells
Applications: Flow Cytometry
Protein signatures for classification and prognosis of gastric cancer a signaling pathway-based approach.
Authors: Wang D, Ye F, Sun Y, Li W, Liu H, Jiang J, Zhang Y, Liu C, Tong W, Gao L, Sun Y, Zhang W, Seetoe T, Lee P, Suo J, Zhang DY
Am. J. Pathol., 2011;179(4):1657-66.
Species: Human
Sample Types: Tissue Homogenates
Applications: Array Development
Combined inhibition of MET and EGFR suppresses proliferation of malignant mesothelioma cells.
Authors: Kawaguchi K, Murakami H, Taniguchi T, Fujii M, Kawata S, Fukui T, Kondo Y, Osada H, Usami N, Yokoi K, Ueda Y, Yatabe Y, Ito M, Horio Y, Hida T, Sekido Y
Carcinogenesis, 2009;30(7):1097-105.
Species: Human
Sample Types: Whole Tissue
Applications: IHC-P
Alternative proteolytic processing of hepatocyte growth factor during wound repair.
Authors: Buchstein N, Hoffmann D, Smola H, Lang S, Paulsson M, Niemann C, Krieg T, Eming SA
Am. J. Pathol., 2009;174(6):2116-28.
Species: Human
Sample Types: Whole Tissue
Applications: IHC-Fr