Product Pathways - PathScan ELISA
PathScan® Total HSP27 Sandwich ELISA Kit #7295
| Kit Includes | Volume | Solution Color |
|---|---|---|
| HSP27 Antibody Coated Microwells | ||
| HSP27 Detection Antibody | 11 milliliters | Green |
| Anti-mouse IgG HRP-Linked Antibody | 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 Mk
Reactivity Key: H=Human Mk=Monkey
Description
CST's PathScan® Total HSP27 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of total HSP27 protein. An HSP27 Antibody 2376* has been coated onto the microwells. After incubation with cell lysates, both nonphospho- and phospho-HSP27 are captured by the coated antibody. Following extensive washing, an HSP27 Mouse mAb #2402* is added to detect the captured HSP27 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 HSP27 protein.* Antibodies in kit are custom formulations specific to kit.
Specificity / Sensitivity
CST's PathScan® Total HSP27 Sandwich ELISA Kit detects endogenous levels of total HSP27 protein. Using PathScan® Phospho-HSP27 (Ser78) Sandwich ELISA Kit #7290, a significant induction of phospho-HSP27 (Ser78) in HeLa cells treated with UV light can be detected. However, the level of total HSP27 (phospho and non-phospho), detected by this Sandwich ELISA Kit #7295, remains unchanged (Figure 1). COS cells treated with UV light show similar results (data not shown).
Western Blotting
Figure 1: Treatment of HeLa cells with UV light stimulates phosphorylation of HSP27 at Ser78, detected by PathScan® Phospho-HSP27 (Ser78) Sandwich ELISA kit, #7290, but does not affect the level of total HSP27 protein detected by PathScan® Total HSP27 Sandwich ELISA kit, #7295. OD450 readings (upper) and the corresponding Western blot using Phospho-HSP27 (Ser78) Ab #2405 (lower right) or HSP27 (G31) Mouse mAb #2402 (lower left), are shown.
Sandwich ELISA
Figure 2: Relationship between protein concentration of lysates from untreated and UV-treated HeLa cells and kit assay optical density readings. HeLa cells (70-85% confluence) were treated with UV and lysed after incubation at 37oC for 30 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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