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.
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.
|Product Includes||Volume||Solution Color|
|HSP27 Mouse mAb Coated Microwells||96 tests|
|Phospho-HSP27 (Ser78) Rabbit Detection Antibody||1 ea||Green (Lyophilized)|
|Anti-rabbit IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
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. Anti-rabbit IgG, HRP-linked antibody 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 in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
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). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
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 HSPs, HSP27 is regulated at both the transcriptional and posttranslational levels (1). In response to stress, the HSP27 expression increases several-fold to confer cellular resistance to the adverse environmental change. HSP27 is phosphorylated at Ser15, Ser78, and Ser82 by MAPKAPK-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).
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