Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Phospho-HSP27 (Ser82) Sandwich ELISA Kit #7152

When ordering five or more kits, please contact us for processing time and pricing at sales@cellsignal.com.

Important Ordering Details: Product is assembled upon order to ensure maximum activity. Domestic: Please allow up to two weeks for your order to be processed and shipped. International: Please allow up to three weeks, depending on the country, for your order to be processed and shipped.

Kit Includes Volume Solution Color
Hsp27 Ab coated microwells 96 tests
Hsp27 (S82) DetectionAb 11 ml Green
Anti-rabbit IgG, HRP-linked Antibody 11 ml Red
TMB Substrate #7004 11 ml Colorless
STOP Solution #7002 11 ml Colorless
Sealing Tape 2 sheets
ELISA Wash Buffer (20X) 25 ml Colorless
ELISA Sample Diluent 25 ml Blue
Cell Lysis Buffer (10X) #9803 15 ml 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, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H

Reactivity Key:  H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

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 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 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.

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. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA - Western correlation

ELISA - Western correlation

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.

Sensitivity

Sensitivity

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 and lysed after incubation at 37C 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 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).

  1. Arrigo, A.P. and Landry, J. (1994) Cold Spring Harbor Laboratory Press, NY, 335-373.
  2. Landry, J. et al. (1992) J. Biol. Chem. 267, 794-803.
  3. Rouse, J. et al. (1994) Cell 78, 1027-1037.
  4. Rogalla, T. et al. (1999) J. Biol. Chem. 274, 18947-18956.
  5. Lavoie, J. et al. (1993) J. Biol. Chem. 268, 24210-24214.
  6. Rousseau, S. et al. (1997) Oncogene 15, 2169-2177.

Application References

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