| | PhosphoDetect™ Hsp27 (pSer78/82) ELISA Kit |
|  | Cat. No. CBA078 |
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| Format: 96-well plate | | Form: 96 Tests | | Detection Method: Colorimetric | | Assay Range: 0.1-10 ng/ml | | Assay Time: 4.5 h | | Sample Type: Cell lysates | | Positive Control: Phosphorylated Hsp27 Standard, UV-treated HeLa or MCF7 cells or sorbitol-treated DU145 cells | | Kit Contents: Hsp27-Coated Plate, Rabbit Anti-Hsp27 Detector Antibody, Phosphorylated Hsp27 Standard, HRP-Conjugate, Assay Diluent, ELISA 20X Plate Wash Concentrate, TMB Substrate, ELISA Stop Solution, and a user protocol | | Comments: Detects and quantifies the level of phosphorylated Hsp27 protein in cell lysates, tissue extracts, and biological fluids. Hsp27 is the small heat shock protein that acts as a molecular chaperone interacting with a large number of proteins. Hsp27 is known for its ability to negatively regulate the mitochondrial pathway to caspase activation. The phosphorylated form of Hsp27 also plays a major role in apoptosis through its ablity to prevent cell death by maintaining redox homeostasis and mitochondrial stability. | | Ref.: Xu, L., et al. 2006. Oncogene 25, 2987. Shin, K.D., et al. 2005. J. Biol. Chem. 280, 41439. Whitesell, L. and Lindquist, S. I. 2005. Nature Rev. 5, 761. Eustace, B.K., et al. 2004. Nature Cell Biol. 6, 507. Bruey, J-M., et al. 2000. Nature Cell Biol. 2, 645. Cornford, P.A., et al. 2000. Cancer Res. 60, 7099. Garrido, C., et al. 1999. FASEB J. 13, 2061. |
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Detection of Phosphorylated Hsp27 by Western Blotting. (A) MCF-7 and HeLa cells were either left untreated (-UV) or exposed to UV-C radiation (+UV). Cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009), subjected to SDS-PAGE, and transferred to nitrocellulose membrane for Western blotting analysis using Anti-Phospho-Hsp27 Detector Antibody Lane 1: MWM; lane 2: MCF-7, -UV; lane 3: MCF-7, +UV; lane 4: HeLa, -UV; lane 5: HeLa, +UV. (B) Unphosphorylated Hsp27 and recombinant Hsp27 that had been phosphorylated in vitro with MAPKAP-2 kinase were subjected to SDS-PAGE and Western blotting analysis using Anti-Phospho-Hsp27 Detector Antibody. Lane 1: MWM; lane 2: in vitro phosphorylated, recombinant Hsp27; lane 3: unphosphorylated, recombinant Hsp27.
Phosphorylation of Hsp27 in the Presence of Inhibitor. MCF7 cells were either left untreated (left bar) or treated with a highly specific, cell-permeable inhibitor of p38 MAP kinase, SB203580 (Cat. No. 559389), (2 µM) followed by UV treatment (right bar). Cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) and the level of phosphorylated Hsp27 was determined as outlined in the Detailed Protocol.
Protocol Summary.
Quantification of Phosphorylated Hsp27 in Tumor Cells. Various tumor cell lines (indicated in the figure) were exposed to 100 J/m2 UV irradiation or treated with 400 mM sorbitol for 30 min. Cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) according to the recommended protocol. The level of phosphorylated Hsp27 was determined as outlined in the Detailed Protocol and expressed as ng phosphorylated Hsp27/mg total protein.
Representative Standard Curve. A standard curve was generated using phosphorylated (black square) and unphosphorylated (black triangle) Hsp27 as outlined in the Detailed Protocol. Assay Range: 0.1-10 ng/ml; lower limit of detection: ~0.1 ng/ml.
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