Figure 1: Constitutive phosphorylation of Met in HCC827 cells lysed in the presence of phosphatase inhibitors (phospho lysate) is detected by PathScan® Phospho-Met (Tyr1349) Sandwich ELISA Kit #7896 (top, right). In contrast, a low level of phospho-Met protein is detected in HCC827 cells lysed without addition of phosphatase inhibitors* (nonphospho lysate). Similar levels of total Met protein from either nonphospho or phospho lysates are detected by PathScan® Total Met Sandwich ELISA Kit #7242 (top, left). Absorbance at 450 nm is shown in the top figure, while the corresponding Western blots using Phospho-Met (Tyr1349) Rabbit mAb #3133 (right) or a total Met Antibody #4560 (left) are shown in the bottom figure. *Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate and Na3VO4.
Figure 2: The relationship between protein concentration of phospho or nonphospho lysates and the absorbance at 450 nm is shown. Unstarved HCC827 cells were cultured (85% confluence) and lysed with or without addition of phosphatase inhibitor (phospho or nonphospho lysate).
The PathScan® Phospho-Met (Tyr1349) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Met when phosphorylated at Tyr1349. A Met rabbit antibody has been coated onto the microwells. After incubation with cell lysates, phospho-Met (Tyr1349) is captured by the coated antibody. Following extensive washing, a biotinylated phospho-Met (Tyr1349) rabbit detection antibody is added to detect the captured phospho-Met protein. HRP-linked streptavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The absorbance at 450nm is proportional to the quantity of Met phosphorylated at Tyr1349.
Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Phospho-Met (Tyr1349) Sandwich ELISA Kit detects endogeneous levels of Met when phosphorylated at Tyr1349 (see Figure 1). The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Met, a high affinity tyrosine kinase receptor for hepatocyte growth factor (HGF, also known as scatter factor) is a disulfide-linked heterodimer made of 45 kDa α- and 145 kDa β-subunits (1,2). The α-subunit and the amino-terminal region of the β-subunit form the extracellular domain. The remainder of the β-chain spans the plasma membrane and contains a cytoplasmic region with tyrosine kinase activity. Interaction of Met with HGF results in autophosphorylation at multiple tyrosines, which recruit several downstream signaling components, including Gab1, c-Cbl, and PI3 kinase (3). These fundamental events are important for all of the biological functions involving Met kinase activity. The addition of a phosphate at cytoplasmic Tyr1003 is essential for Met protein ubiquitination and degradation (4). Phosphorylation at Tyr1234/1235 in the Met kinase domain is critical for kinase activation. Phosphorylation at Tyr1349 in the Met cytoplasmic domain provides a direct binding site for Gab1 (5). Research studies have shown that altered Met levels and/or tyrosine kinase activities are found in several types of tumors, including renal, colon, and breast. Thus, investigators have concluded that Met is an attractive potential cancer therapeutic and diagnostic target (6,7).
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