Cell Signaling Technology

Product Pathways - Tyrosine Kinase / Adaptors

PathScan® Total Met Sandwich ELISA Kit #7242

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Kit Includes Volume Solution Color
Met mAb coated microwells 96 tests
Met Detection Ab 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Sealing Tape 2 sheets
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

CST's PathScan® Total Met Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Met protein. A Met Mouse mAb has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-Met proteins are captured by the coated antibody. Following extensive washing, Met Rabbit Antibody is added to detect both the captured phospho- and nonphospho-Met protein. Anti-rabbit IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of total Met protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Total Met Sandwich ELISA Kit #7242 detects endogenous levels of total Met protein. As shown in Figure 1, bothphospho- and nonphospho-Met proteins from untreated and HGF-treated A431 cell lysates are detected by this kit. In Figure 3, Western blot analysis of protein captured in the Met Mouse mAb coated microwell shows a single band corresponding to the Met protein. 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: Non-phospho and phospho Met proteins from untreated and HGF-treated A431 cells can be detected by PathScan® Total Met Sandwich ELISA kit #7242 with similar optical density readings. OD 450 readings are shown in the top figure, while the corresponding Western blots using Met Mouse mAb #3127 (left panel) or Met (Tyr1234/1235) Rabbit mAb #3129 (right panel), are shown in the bottom figure.

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of lysates from untreated and HGF-treated A431 cells and kit assay optical density readings is shown. After starvation, A431 cells (85% confluence) were treated with HGF (40 ng/ml) for 5 min at 37°C, and then lysed.

Specificity

Specificity

Figure 3: Kit specificity demonstrated by Western blot analysis of the ELISA-well captured protein is shown. Lysates were prepared from human A431 cells and incubated in wells coated with a Met Mouse mAb. Wells were then washed, and captured protein was solubilized in SDS gel loading buffer. A431 lysate (lane 1) and captured protein (lane 2) were analyzed by Western blot using a Met Rabbit antibody. A single band corresponding to the Met protein is detected in the captured material (lane 2).


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.

Application References

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

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