Product Pathways - PathScan ELISA
PathScan® Total c-Jun Sandwich ELISA Kit II #7150
| Kit Includes | Volume | Solution Color |
|---|---|---|
| c-Jun Rabbit mAb coated Microwells | ||
| c-Jun 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 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 M R
Reactivity Key: H=Human M=Mouse R=Rat
Description
CST's PathScan® Total c-Jun Sandwich ELISA Kit II is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total c-Jun protein. A c-Jun rabbit mAb (#2374*) has been coated onto the microwells. After incubation with cell lysates, c-Jun protein is captured by the coated antibody. Following extensive washing, c-Jun Mouse mAb (#2379*) is added to detect the captured c-Jun protein. HRP-linked anti-mouse 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 c-Jun protein.* Antibodies in this kit are custom formulations specific to the kit.
Specificity / Sensitivity
CST's PathScan® Total c-Jun Sandwich ELISA Kit II detects endogenous levels of total c-Jun protein. As shown in Figure 1, using PathScan® Phospho-c-Jun (Ser63) Sandwich ELISA Kit II #7145, a significant induction of phospho-c-Jun (Ser63) in UV-treated 293 cells can be detected. However, the level of total c-Jun protein (phospho and non-phospho), detected by this Sandwich ELISA Kit #7150, remains unchanged. Both C6 and NIH/3T3 cells treated with UV light or anisomycin show similar results (data not shown).This kit is more sensitive than kit #7270 (figure 2).
Western Blotting
Figure 1: Treatment of 293 cells with UV stimulates phosphorylation of c-Jun at Ser63, detected by PathScan® Phospho-c-Jun (Ser63) Sandwich ELISA Kit II #7145, but does not affect the level of total c-Jun protein detected by PathScan® Total c-Jun Sandwich ELISA Kit II #7150. Lambda protein phosphatase (LPP) treatment of control cell lysates (37°C for 90 minutes) abolished the basal phosphorylation of c-Jun in control lysates shown in both Sandwich ELISA and Western analysis. The OD450 readings are shown in the top figure, while the corresponding Western blot, using Phospho-c-Jun (Ser63) rabbit mAb #2378 (right panel) or c-Jun rabbit mAb (6H5) #2374 (left panel), is shown in the bottom figure.
Sandwich ELISA
Figure 2: Linear relationship between protein concentration of lysates from UV-treated 293 cells and kit assay optical density readings. Pathscan® Total c-Jun Sandwich ELISA Kit II #7150 is more sensitive than kit #7270 over a range of lysate concentrations. 293 cells (70-90% confluence) were treated without or with UV and lysed after incubation at 37oC for 30 minutes.
Background
c-Jun is a component of the transcription factor AP-1, which binds and activates transcription at TRE/AP-1 elements. The transcriptional activity of c-Jun is regulated by phosphorylation at Ser63 and Ser73 (1,2). Extracellular signals including growth factors, transforming oncoproteins and UV irradiation stimulate phosphorylation of c-Jun at Ser63/73 and activate c-Jun-dependent transcription. Mutation of Ser63/73 renders c-Jun nonresponsive to mitogenic and stress-induced signaling pathways. The MAP kinase homologue SAPK/JNK binds to the amino-terminal region of c-Jun and phosphorylates c-Jun at Ser63/73. In addition, the activity of SAPK/JNK is stimulated by the same signals that activate c-Jun (3,4).
- Binetruy, B. et al. (1991) Nature 351, 122-127.
- Smeal, T. et al. (1991) Nature 354, 494-496.
- Derijard, B. et al. (1994) Cell 76, 1025-1037.
- Kyriakis, J.M. et al. (1994) Nature 369, 156-160.
Application References
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Companion Products
Rabbit Monoclonals Produced UsingTechnology from Epitomics, Inc. UnderU.S. patent No. 5,675,063.
