A solid phase sandwich enzyme-linked immunosorbent assay (ELISA). Standard dilutions and samples are added to the wells precoated with Anti-HSP27 capture antibody, specific for HSP27 regardless of phosphorylation state. After 2 hr at RT, a detection antibody specific for the non-phosphorylated or phosphorylated protein is added and incubated 1 hr at RT, which results in binding to the immobilized HSP27 protein. An anti-rabbit IgG-HRP binds to the immobilized protein completing the four-member sandwich. The reaction is visualized by TMB substrate, followed by the stop solution. The intensity of the yellow color, measured in a multiwell plate reader at 450 nm, is directly proportional to the concentration of HSP27 in original sample. The unknown concentrations are calculated from the standard curve run with each assay.
Phospho-Heat Shock Protein 27 pSer82 ELISA is designed to detect and quantify the level of HSP27 protein, phosphorylated on serine 82. This assay is intended for the detection of Phospho-HSP27 pSer82 in lysates of human cells. It does not react with the mouse or rat HSP27 homolog, HSP25. For normalizing the HSP27 content of the samples, Heat Shock Protein 27 ELISA, which permits quantification of HSP27 independent of phosphorylation status, is available from Sigma (Product No. CS0640).
HSP27, a member of the ubiquitously expressed anti-apoptotic heat shock protein group, plays a role in maintaining cellular redox by increasing glutathione levels, stabilizing actin filaments, and blocking actin polymerization. It prevents the aggregation of improperly folded proteins. Recent evidence suggests that HSP27 protects cells from apoptosis by participation in several signaling pathways.
The sensitivity is <1.0 units/mL, which is 4 times more than immunoblotting. The specificity is confirmed by peptide competition. Only the phosphopeptide blocks the ELISA signal.