Product Pathways - PathScan ELISA
PathScan® Phospho-Met (panTyr) Sandwich ELISA Kit #7333
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Met Rabbit Antiboby Coated Microwells | 96 tests | |
| Phospho-Tyrosine Mouse Detection Antibody | 11 milliliters | Green |
| Anti-mouse IgG, HRP-Linked Antibody | 11 milliliters | Red |
| TMB Substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| Sealing Tape | 2 sheets | |
| 20X Wash Buffer | 25 milliliters | Colorless |
| Sample Diluent | 25 milliliters | Blue |
| 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
The PathScan® Phospho-Met (panTyr) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of Met when tyrosine phosphorylated. A Met Rabbit Antibody* has been coated onto the microwells. After incubation with cell lysates, Met (phospho and nonphospho) is captured by the coated antibody. Following extensive washing, a Phospho-Tyrosine Mouse Detection Antibody* is added to detect tyrosine phosphorylation of the captured 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 on tyrosine.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Phospho-Met (panTyr) Sandwich ELISA Kit #7333 detects Met when tyrosine phosphorylated. High levels of phospho-Met protein are detected in HCC827 cells where Met is constitutively phosphorylated (Figure 1). 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.
Sandwich ELISA
Figure 1: Constitutive phosphorylation of Met in HCC827 cells lysed in the presence of phosphatase inhibitors (phospho lysate) is detected by PathScan® Phospho-Met (panTyr) Sandwich ELISA Kit #7333 (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.
Sandwich ELISA
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. 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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Companion Products
- 7242 PathScan® Total Met Sandwich ELISA Kit
- 3135 Phospho-Met (Tyr1003) (13D11) Rabbit mAb
- 3129 Phospho-Met (Tyr1234/1235) (3D7) Rabbit mAb
- 3126 Phospho-Met (Tyr1234/1235) Antibody
- 3133 Phospho-Met (Tyr1349) (130H2) Rabbit mAb
- 3121 Phospho-Met (Tyr1349) Antibody
- 4560 Met Antibody
- 3127 Met (25H2) Mouse mAb
- 9803 Cell Lysis Buffer (10X)
- 7076 Anti-mouse IgG, HRP-linked Antibody
