Product Pathways - Tyrosine Kinase/ Adaptors
PathScan® Total Met Sandwich ELISA Kit #7242
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Met Mouse mAb coated microwells | 96 tests | |
| Met Detection Ab | 11 milliliters | Green |
| Anti-rabbit IgG HRP-Linked Ab | 11 milliliters | Red |
| TMB Substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| 20X Wash Buffer | 25 milliliters | Colorless |
| Sample Diluent | 25 milliliters | Blue |
| Sealing Tape | 2 sheets | |
| Cell Lysis Buffer (10X) # 9803 | 15 milliliters | 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 (10X), which is stored at –20°C (packaged separately).
Species Cross-Reactivity
H
Reactivity Key: H=Human
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 #3127* 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 #3123* is added to detect both the captured phospho- and nonphospho-Met protein. Anti-rabbit IgG, HRP-linked Antibody #7074* 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 #3127 coated microwell shows a single band corresponding to the Met protein.
Western Blotting
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 capture antibody #3127. 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 Met antibody #3123. A single band corresponding to the Met protein is detected in the captured material (lane 2).
Sandwich ELISA
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.
Sandwich ELISA
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.
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. Addition of a phosphate at cytoplasmic Tyr1003 is essential for ubiquitination and Met protein degradation (4). Phosphorylation of Tyr1234/1235 in the Met kinase domain is critical to kinase activation. Phosphorylation of Tyr1349 in the Met cytoplasmic domain provides a direct binding site for Gab1 (5). Altered Met levels and/or tyrosine kinase activities are found in several types of tumors, including renal, colon and breast cancers. Thus, Met is an attractive cancer therapeutic and diagnostic target (6).
- Weidner, K.M. et al. (1993) J. Cell Biol. 121, 145-154.
- Park, M. et al. (1986) Cell 45, 895-904.
- Bardelli, A. et al. (1997) Oncogene 15, 3103-3111.
- Taher, T.E. et al. (2002) J. Immunol. 169, 3793-3800.
- Schaeper, U. et al. (2000) J. Cell Biol. 149, 1419-1432.
- Traxler, P. et al. (2001) Med. Res. Rev. 21, 499-512.
Application References
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