Nori Mouse C-Met ELISA Kit Summary

Alternative names for C-Met: tyrosine-protein kinase Met,  hepatocyte growth factor receptor, HGFR, proto-oncogene c-Met

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

c-Met, also called tyrosine-protein kinase Met or hepatocyte growth factor receptor (HGFR),^[1] is a protein that is encoded by the MET gene. The protein possesses tyrosine kinase activity. MET is a receptor tyrosine kinase that is produced as a single-chain precursor. The precursor is proteolytically cleaved at a furin site to yield a highly glycosylated extracellular α-subunit and a transmembrane β-subunit, which are linked together by a disulfide bridge.^[2] MET is essential for embryonic development, organogenesis and wound healing. HGF/Scatter Factor (SF) and its splicing isoform (NK1, NK2) are the only known ligands of the MET receptor. MET is normally expressed by cells of epithelial origin, while expression of HGF/SF is restricted to cells of mesenchymal origin. When HGF/SF binds its cognate receptor MET it induces its dimerization through a not yet completely understood mechanism leading to its activation. MET activation by its ligand HGF induces MET kinase catalytic activity, which triggers transphosphorylation of the tyrosines Tyr 1234 and Tyr 1235. These two tyrosines engage various signal transducers,^[3] thus initiating a whole spectrum of biological activities driven by MET, collectively known as the invasive growth program. The transducers interact with the intracellular multi-substrate docking site of MET either directly, such as GRB2, SHC,^[4] SRC, and the p85 regulatory subunit of phosphatidylinositol-3 kinase,^[5] or indirectly through the scaffolding protein Gab1^[6] Abnormal MET activation in cancer correlates with poor prognosis, where aberrantly active MET triggers tumor growth, formation of new blood vessels that supply the tumor with nutrients, and cancer spread to other organs. MET is deregulated in many types of human malignancies, including cancers of kidney, liver, stomach, breast, and brain. Normally, only stem cells and progenitor cells express MET, which allows these cells to grow invasively in order to generate new tissues in an embryo or regenerate damaged tissues in an adult. However, cancer stem cells are thought to hijack the ability of normal stem cells to express MET, and thus become the cause of cancer persistence and spread to other sites in the body. Both the overexpression of Met/HGFR, as well as its autocrine activation by co-expression of its hepatocyte growth factor ligand, have been implicated in oncogenesis.^[3]

References

1. Bottaro DP, et al. (1991). Science. 251 (4995): 802–4.
2. Birchmeier C, et al. ().Nat. Rev. Mol. Cell Biol. 4 (12): 915–25.
3. Johnson M, et al. (1995). Biochemistry and Molecular Biology International. 36 (3): 465–74.
4. Pelicci G, et al. (1995). Oncogene. 10 (8): 1631–8.
5. Weidner KM, et al. (1996). Nature. 384 (6605): 173–6.
6. Weidner KM, et al. (1996). Nature. 384 (6605): 173–6.