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.
Figure 2: The relationship between protein concentration of lysates from untreated and Forskolin treated COS cells and kit assay optical density readings is shown. COS cells were treated with Forskolin #3828 (50 µM) for 30 min at 37°C, and then lysed.
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).
|REACTIVITY||H M Mk|
|Product Includes||Volume||Solution Color|
|β-Catenin Mouse mAb Coated Microwells||96 tests|
|B-Catenin 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|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
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 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 rabbit mAb is added to detect both the captured phospho- and nonphospho-Beta-catenin 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 Beta-catenin 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 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. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.Species Reactivity:
Human, Mouse, Monkey
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 at Ser45. This phosphorylation event primes β-catenin for subsequent phosphorylation by GSK-3β (4-6). GSK-3β destabilizes β-catenin by phosphorylating it at Ser33, Ser37, and Thr41 (7). Mutations at these sites result in the stabilization of β-catenin protein levels and have been found in many tumor cell lines (8).
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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.