Product Pathways - PathScan ELISA
PathScan® Phospho-cdc2 (Thr161) Sandwich ELISA Kit #7184
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Cdc2 (Thr161) Ab Coated Microwells | 96 tests | |
| Cdc2 Detection Antibody | 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 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
CST's PathScan® Phospho-cdc2 (Thr161) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-cdc2 (Thr161) protein. A Phospho-cdc2 (Thr161) Rabbit polyclonal Ab #9114* has been coated onto the microwells. After incubation with cell lysates, phospho-cdc2 (Thr161) protein is captured by the coated antibody. Following extensive washing, cdc2 Mouse mAb #2658* is added to detect the captured phospho-cdc2 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 phospho-cdc2 (Thr161) protein.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Phospho-cdc2 (Thr161) Sandwich ELISA Kit detects endogenous levels of phospho-cdc2 (Thr161). As shown in Figure 1, using the Phospho-cdc2 (Thr161) ELISA Kit #7184, a significant induction of phospho-cdc2 (Thr161) is detected in HeLa cells treated with Interleukin-4.
Sandwich ELISA
Figure 2: The relationship between protein concentration of lysates from untreated and Interleukin-4 treated HeLa cells and kit assay optical density reading. HeLa cells (80% confluent) were treated with human Interleukin-4 for 10 minutes (100 ng/ml).
Sandwich ELISA
Figure 1: Treatment of HeLa cells with Interleukin-4 stimulates phosphorylation of cdc2 at Thr161 as detected by PathScan® Phospho-cdc2 (Thr161) Sandwich ELISA kit #7184, but does not affect the level of total cdc2 protein detected using cdc2 Mouse mAb (#9116) via Western analysis. OD 450 readings are shown in the top figure, while the corresponding Western blot using Phospho-cdc2 (Thr161) Antibody #9114 (left panel) or cdc2 Mouse mAb #9116 (right panel), is shown in the bottom figure.
Background
The entry of eukaryotic cells into mitosis is regulated by cdc2 kinase activation, a process controlled at several steps including cyclin binding and phosphorylation of cdc2 at Thr161 (1). However, the critical regulatory step in activating cdc2 during progression into mitosis appears to be dephosphorylation of cdc2 at Tyr15 and Thr14 (2). Phosphorylation at Thr14 and Tyr15 resulting in inhibition of cdc2 can be carried out by Wee1 and Myt1 protein kinases (3,4). The cdc25 phosphatase may be responsible for removal of phosphates at Thr14 and Tyr15 and subsequent activation of cdc2 (1,5).
- Atherton-Fessler, S. et al. (1994) Mol. Biol. Cell. 5, 989-1001.
- Norbury, C. et al. (1991) EMBO. J. 10, 3321-3329.
- McGowan, C.H. and Russell, P. (1993) EMBO J. 12, 75-85.
- Wells, N.J. et al. (1999) J. Cell. Sci. 112, 3361-3371.
- Hunter, T. (1995) Cell 80, 225-236.
Application References
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