Cell Signaling Technology

Product Pathways - PathScan ELISA

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

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Kit Includes Volume Solution Color
P-Met (Y1234/1235) AbCoated Microwells 96 tests
Met Detection Ab 11 ml Green
Anti-mouse IgG, HRP-linked Antibody 11 ml Red
STOP Solution #7002 11 ml Colorless
TMB Substrate #7004 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml Yellowish

Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

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 kit are custom formulations specific to kit.

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.

ELISA - Western correlation

ELISA - Western correlation

Figure 1: Constitutive phosphorylation of Met in HCC827 cells lysed in the presence of phosphatase inhibitors (phospho lysate) is detected by PathScan® Phospho-Met (Tyr1234/1235) Sandwich ELISA Kit #7227 (top, right). In contrast, a low level of phospho-Met protein is detected in HCC827 cells lysed without addition of phosphatase inhibitors to the lysis buffer (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 (Tyr1234/1235) Antibody #3126 (right) or a total Met Antibody #4560 (left) are shown in the bottom figure.

Sensitivity

Sensitivity

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 to the lysis buffer (phospho or nonphospho lysate).

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).

  1. Cooper, C.S. et al. (1984) Nature 311, 29-33.
  2. Bottaro, D.P. et al. (1991) Science 251, 802-4.
  3. Bardelli, A. et al. (1997) Oncogene 15, 3103-11.
  4. Taher, T.E. et al. (2002) J Immunol 169, 3793-800.
  5. Schaeper, U. et al. (2000) J Cell Biol 149, 1419-32.
  6. Eder, J.P. et al. (2009) Clin Cancer Res 15, 2207-14.
  7. Sattler, M. and Salgia, R. (2009) Update Cancer Ther 3, 109-118.

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