The Human c-Met (Soluble) ELISA research-use-only kit is designed to detect and quantify the level of soluble human c-Met protein in serum, plasma, and cell culture supernatants using 96-well plates and a microplate reader. The assay will recognize both natural and recombinant soluble human c-Met.
Performance characteristics • Sensitivity: <0.5 ng/mL • Standard curve range: 0.78–50 ng/mL • Sample types: serum, plasma, cell culture supernatants • Species cross-reactivity: human • Sample volume: 100 μL (1:2 to 1:100) • Total assay incubation time: 4 hrs
Principle of the method The Human c-Met (Soluble) kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA). A highly purified antibody has been coated onto the wells of the microtiter strips provided. During the first incubation, standards of known soluble human c-Met content, controls, and unknown samples are pipetted into the wells. After washing, biotinylated secondary antibody is added. After washing, streptavidin-peroxidase (enzyme) is added. This binds to the biotinylated antibody to complete the four-member sandwich. After a third incubation and washing to remove all the unbound enzyme, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of soluble human c-Met present in the original specimen.
Background information c-Met, a member of the tyrosine kinase superfamily, is the receptor for hepatocyte growth factor, also known as scatter factor (HGF/SF). Cells expressing c-Met include epithelial cells, endothelial cells, blood cells of various types, and glomerular mesenchymal cells. Sources of HGF/SF include mesenchymal cells, mesanglial cells, endothelial cells, macrophages, and tumor cells.
HGF/SF binding to c-Met stimulates receptor dimerization and the phosphorylation of numerous residues within the receptor’s cytoplasmic domain. Phosphorylation of tyrosines 1234 and 1235 of c-Met is required for activation of the receptor’s tyrosine kinase activity. c-Met phosphorylation also generates docking sites for numerous signaling molecules and stimulates receptor internalization via clathrin-coated vesicles. Signaling proteins that are phosphorylated and/or localized in response to c-Met phosphorylation include: Grb2, Shc, Cbl, Crk, cortactin, paxillin, GAB1, PI-3 K, FAK, Src, Ras, ERK1 and 2, JNK, PLC-γ, AKT, and STAT3. HGF/SF stimulation of c-Met expressing cells enhances proliferation, migration, morphogenesis, and protease synthesis, characteristics that are associated with invasive cell phenotype.
Soluble c-Met is a truncated form of the c-Met membrane receptor. It can be cleaved by proteases and released from the lipid bilayer in a process known as ectodomain shedding. Many transmembrane proteins are released through this shedding process and it is a normal part of development which when defective can cause a number of pathologies. The soluble form of the c-Met receptor is smaller than the membrane bound receptor, contains the extracellular region of the receptor, and is able to bind the HGF ligand.
Detect low-expressing proteins and use less sample ELISA kits designed to measure intracellular signaling targets are typically 2–10 times more sensitive than western blotting. The improved sensitivity enables you to detect low-expressing proteins that otherwise may not be distinguishable from background. In addition, the amount of sample needed to run the assay is less than what is needed for western blots.
Each ELISA kit is validated for sensitivity, specificity, precision, and lot-to-lot consistency. See product insert for more information on validation.
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