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.
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.
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).
|Product Includes||Volume||Solution Color|
|Met Mouse mAb Coated Microwells||96 tests|
|Met Rabbit Detection mAb||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
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.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Total Met Sandwich ELISA Kit #7242 detects endogenous levels of total Met protein. As shown in Figure 1, both
phospho- 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.
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).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.