Product Pathways - PathScan ELISA
PathScan® Total β-Catenin Sandwich ELISA Kit #7308
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Beta-Catenin Antibody coated microwells | 96 tests | |
| Beta-Catenin Detection Ab | 11 milliliters | Green |
| Anti-mouse IgG HRP-linked Ab | 11 milliliters | Red |
| TMB substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| Sealing Tape | 2 sheets | |
| 20X Washing 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 M
Reactivity Key: H=Human M=Mouse
Description
CST's PathScan® Total Beta-Catenin Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total Beta-catenin protein. A Beta-Catenin Ab #9562* has been coated onto the microwells. After incubation with cell lysates, both phospho- and nonphospho-Beta-catenin proteins are captured by the coated antibody. Following extensive washing, Beta-Catenin Mouse mAb #2322* is added to detect both the captured phospho- and nonphospho-Beta-catenin protein. Anti-mouse IgG, HRP-linked Antibody #7076* 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 Beta-catenin protein.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Total Beta-Catenin Sandwich ELISA Kit #7308 detects endogenous levels of total beta-catenin protein. As shown in Figure 1, both phospho-and nonphospho-β-catenin protein from untreated and Calyculin A-treated 293 cell lysates are detected by this kit. In Figure 3, Western blot analysis of protein captured in the β-Catenin Antibody #9562 coated microwell shows a major band corresponding to the β-catenin protein.
Western Blotting
Figure 3: Kit specificity demonstrated by Western blot analysis of the ELISA-well captured protein. Lysates were prepared from human 293 cells and incubated in wells coated with capture β-Catenin Antibody #9562. Wells were then washed and captured protein was solubilized in SDS gel loading buffer. Western blot analysis of 293 cell starting lysate (lane 1) and captured protein (lane 2) was performed, using β-Catenin Mouse mAb #2322. The major band detected in the captured material corresponds to the β-catenin protein (lane 2).
Sandwich ELISA
Figure 2: The relationship between protein concentration of lysates from untreated and Calyculin A-treated 293 cells and kit assay optical density readings is shown. 293 cells were treated with Calyculin A #9902 (50 nM) for 10 min at 37?C, and then lysed.
Sandwich ELISA
Figure 1: Nonphospho- and phospho-β-catenin proteins from untreated and Calyculin A-treated 293 cells can be detected by PathScan® Total β-Catenin Sandwich ELISA kit #7308 with similar optical density readings. OD 450 readings are shown in the top figure, while the corresponding Western blot using Phospho-β-Catenin (Ser45) Antibody #9564 (right panel) or β-Catenin Antibody #9562 (left panel), is shown in the bottom figure.
Background
β-catenin is a key downstream effector in the Wnt signaling pathway (1). It is implicated in two major biological processes in vertebrates: early embryonic development (2) and tumorigenesis (3). CK1 phosphorylates β-catenin on Ser45. This phosphorylation event primes β-catenin for subsequent phosphorylation by GSK-3 (4-6). GSK-3β destabilizes β-catenin by phosphorylating it at Ser33, Ser37 and Thr41 (7). Mutations in these phosphorylation sites, which result in the stabilization of β-catenin protein levels, have been found in many tumor cell lines (8).
- Cadigan, K.M. and Nusse, R. (1997) Genes Dev. 11, 3286-3305.
- Wodarz, A. and Nusse, R. (1998) Annu. Rev. Cell. Dev. Biol. 14, 59-88.
- Polakis, P. (1999) Curr. Opin. Genet. Dev. 9, 15-21.
- Amit, S. et al. (2002) Genes Dev. 16, 1066-1076.
- Lin, C. et al. (2002) Cell 108, 837-847.
- Yanagawa, S. et al. (2002) EMBO J. 21, 1733-1742.
- Yost, C. et al. (1996) Genes Dev. 10, 1443-1454.
- Morin, P.J. (1997) Science 275, 1787-1790.
Application References
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