Product Pathways - PathScan ELISA
PathScan® Phospho-HSP27 (Ser82) Sandwich ELISA Kit #7152
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Hsp27 Ab coated microwells | 96 tests | |
| Hsp27 (Ser82) Detection Antibody | 11 milliliters | Green |
| Anti-rabbit 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-HSP27 (Ser82) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-HSP27 (Ser82) protein. An Hsp27 Mouse mAb #2402* has been coated onto the microwells. After incubation with cell lysates, HSP27 protein is captured by the coated antibody. Following extensive washing, Phospho-HSP27 (Ser82) Rabbit Antibody #2323* is added to detect the captured phospho-HSP27 (Ser82) protein. Anti-rabbit IgG, HRP-linked 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 (Ser82) protein.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Phospho-HSP27 (Ser82) Sandwich ELISA Kit detects endogenous levels of phospho-HSP27 (Ser82) protein. Using this Sandwich ELISA Kit #7152, a significant induction of phospho-HSP27 (Ser82) can be detected in HeLa cells treated with UV light.
Western Blotting
Figure 1: Treatment of HeLa cells with UV light stimulates phosphorylation of HSP27 at Ser82 detected by PathScan® Phospho-HSP27 (Ser82) Sandwich ELISA Kit #7152. The corresponding Western blots, using Phospho-HSP27 (Ser82) Antibody #2401 (left) or HSP27 (G31) Mouse mAb #2402 (right), are also shown.
Sequence
Figure 2: The relationship between protein concentration of lysates from UV-treated or untreated HeLa cells and kit assay optical density readings. HeLa cells (70-85% confluent) were treated with UV light for 20 minutes.
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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