Product Pathways - PathScan ELISA
PathScan® Phospho-HSP27 (Ser78) Sandwich ELISA Kit #7290
| Kit Includes | Volume | Solution Color |
|---|---|---|
| HSP27 (G31) mAb Coated Microwells | ||
| Phospho-HSP27 (Ser78) Antibody | 11 milliliters | |
| Anti-rabbit IgG, HRP-Linked Antibody | 11 milliliters | |
| 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 Mk
Reactivity Key: H=Human Mk=Monkey
Description
CST's PathScan® Phospho-HSP27 (Ser78) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-HSP27 (Ser78) protein. HSP27 Mouse mAb has been coated onto the microwells. After incubation with cell lysates, HSP27 protein (phosphorylated and nonphosphorylated) is captured by the coated antibody. Following extensive washing, Phospho-HSP27 (Ser78) Antibody is added to detect the captured phospho- HSP27 (Ser78) protein. HRP-linked anti-rabbit antibody #7074* 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- HSP27 (Ser78) protein.* Antibodies are custom formulations specific to the kit.
Specificity / Sensitivity
CST's PathScan® Phospho-HSP27 (Ser78) Sandwich ELISA Kit detects endogenous levels of phospho-HSP27 (Ser78) protein. Using this Sandwich ELISA Kit #7290, a significant induction of phospho-HSP27 (Ser78) can be detected in HeLa cells treated with UV light. However, the level of total HSP27 (phospho and non-phospho), detected by the Total HSP27 Sandwich ELISA Kit #7295, remains unchanged (Figure 1). COS cells treated with UV light show similar results (data not shown).
Sandwich ELISA
Figure 2: Linear relationship between protein concentration of lysates from UV-treated Hela cells and kit assay optical density readings. HeLa cells (70-85% confluent) were treated with ultraviolet light and lysed after growth at 37oC for 30 minutes.
Sandwich ELISA
Figure 1: Treatment of HeLa cells with UV light stimulates phosphorylation of HSP27 at Ser78 detected by PathScanTM Phospho-HSP27 (Ser78) Sandwich ELISA kit, #7290, but does not affect the level of total HSP27 protein detected by PathScanTM Total HSP27 Sandwich ELISA kit, #7295. The corresponding Western blots, using Phospho-HSP27 (Ser78) Antibody #2405 (right) or HSP27 (G31) Monoclonal Antibody #2402 (left), are also shown.
Background
Heat shock protein (HSP) 27 is one of the small HSPs that are constitutively expressed at different levels in various cell types and tissues. Like other small heat shock proteins, HSP27 is regulated at both the transcriptional and posttranslational levels (1). In response to stress, the expression level of HSP27 increases several-fold to confer cellular resistance to the adverse environmental change. HSP27 is phosphorylated at Ser15, Ser78 and Ser82 by MAPKAP kinase 2 as a result of the activation of the p38 MAP kinase pathway (2,3). Phosphorylation of HSP27 causes a change in its tertiary structure, which shifts from large homotypic multimers to dimers and monomers (4). It has been shown that phosphorylation and increased concentration of HSP27 modulates actin polymerization and reorganization (5,6).
- Arrigo, A.P. and Landry, J. (1994) Cold Spring Harbor Laboratory Press, NY, 335-373.
- Landry, J. et al. (1992) J. Biol. Chem. 267, 794-803.
- Rouse, J. et al. (1994) Cell 78, 1027-1037.
- Rogalla, T. et al. (1999) J. Biol. Chem. 274, 18947-18956.
- Lavoie, J. et al. (1993) J. Biol. Chem. 268, 24210-24214.
- Rousseau, S. et al. (1997) Oncogene 15, 2169-2177.
Application References
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