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PathScan^® Phospho-Met (Tyr1234/1235) Sandwich ELISA Kit

PathScan^® Phospho-Met (Tyr1234/1235) Sandwich ELISA Kit #7227

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ELISA

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Supporting Data

REACTIVITY

Application Key:

ELISA-ELISA

Species Cross-Reactivity Key:

H-Human

Product Information

Product Description

The PathScan^® Phospho-Met (Tyr1234/1235) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Met when phosphorylated at Tyr1234/1235. A Phospho-Met (Tyr1234/1235) Rabbit Antibody has been coated onto the microwells. After incubation with cell lysates, phospho-Met (Tyr1234/1235) is captured by the coated antibody. Following extensive washing, a Met Mouse Detection Antibody is added to detect the captured phospho-Met protein. Anti-mouse IgG, HRP-Linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of Met phosphorylated at Tyr1234/1235.

*Antibodies in this kit are custom formulations specific to kit.

Protocol

Specificity / Sensitivity

CST's PathScan^® Phospho-Met (Tyr1234/1235) Sandwich ELISA Kit #7227 detects Met when tyrosine phosphorylated at Tyr1234/1235. As shown in Figure 1, high levels of phospho-Met protein at Tyr1234/1235 are detected in HCC827 cells where Met is constitutively phosphorylated. These high levels are abolished in HCC827 cells lysed without addition of phosphatase inhibitors* to the lysis buffer. The levels of total Met protein (phospho and nonphospho) as detected by PathScan^® Total Met Sandwich ELISA Kit #7242 remain unchanged (Figure 1).
* Phosphatase inhibitors include sodium pyrophosphate, β-glycerophosphate and Na3VO4.
This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

Species Reactivity:

Human

Background

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).

Database Links

UniProt ID: P08581

Entrez-Gene Id: 4233

Limited Uses

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For Research Use Only. Not For Use In Diagnostic Procedures.

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PathScan® Phospho-Met (Tyr1234/1235) Sandwich ELISA Kit #7227