YOUR CART
In response to stress, the expression level of HSP27 increases several fold to confer cellular resistance to the adverse environmental change. HSP27 is localized to the cytoplasm of unstressed cells but can redistribute to the nucleus in response to stress, where it may function to stabilize DNA and/or the nuclear membrane. HSP27 is phosphorylated at serines 15, 78 and 82 by MAPKAPK-2 as a result of the activation of the p38 MAPK pathway. Phosphorylation of HSP27 causes a change in its tertiary structure, which shifts from large homotypic multimers to dimers and monomers. Thus, phosphorylated HSPs have significantly decreased ability to act as molecular chaperones. Phosphorylation of HSP27 on Ser82 by MAPKAPK-2 or AKT leads to HSP dissociation from the AKT/MAPKAPK2/p38 complex, which may promote independent survival signals. The phosphorylation of HSP27 also enhances its association with IKKβ to result in decreased IKK activity.
The FACE™ Method
In FACE, cells are cultured in 96-well plates and stimulated to induce the pathway of interest. Following stimulation, the cells are fixed rapidly, which preserves activation-specific protein modifications. Each well is then incubated with a primary antibody specific for the activated protein of interest. Subsequent incubation with secondary HRP-conjugated antibody and developing solution provides a colorimetric or chemiluminescent readout that is quantitative and reproducible (Figure 1). The number of cells in each well can be normalized easily with the provided Crystal Violet solution. FACE Kits also contain primary antibody specific for the native inactive protein, so you can monitor both native and activated protein levels in the same experiment. FACE eliminates cellular extractions, radioactive kinase assays, time-consuming Westerns and inefficient epitope interactions that occur on membranes. FACE is a highly sensitive high-throughput assay designed for detecting activated proteins within mammalian cells.
