Introducing the PhosphoDetect™ Hsp27 (pSer78/82) ELISA Kit
Heat shock proteins (Hsp) comprise a group of highly conserved proteins that protect cells against different types of physiological and environmental stress. As a member of the small Hsp (sHsp) family of heat shock proteins, Hsp27 plays a role in the regulation of cell survival. The human Hsp27 (pSer78/82) ELISA is a complete kit for the quantitative determination of phosphorylated Hsp27 ( Ser78 and Ser82) in cell lysates, tissue extracts, and biological fluids.
The PhosphoDetect™ Hsp27 (pSer78/82) ELISA kit (Cat. No. CBA078) is:
Highly sensitive: detects Hsp27 at 0.1-10 ng/ml
Quantitative : includes recombinant phosphorylated Hsp27 for standard curve comparisons
Versatile: quantitates human phosphorylated Hsp27 in cell lysates, tissue extracts, and biological fluids
Convenient : 96-well, colorimetric detection
Rollover a description below to view appropriate figure.
Figure 1: 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.
Figure 2: 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.
Figure 3: 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.
Figure 4: 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 mM) 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.
