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MEK1 and MEK2, also called MAP kinase kinase and ERK activator kinase, are dual-specificity protein kinases that function in a mitogen activated protein kinase cascade controlling both cell growth and differentiation. MEK1/2 is activated by a wide variety of growth factors and cytokines, and also by membrane depolarization and calcium influx, with the predominant activators being Raf kinases. MEK is involved in the MAPK signaling pathway of Ras-Raf-MEK-ERK. This signaling cascade is an important target in cancer therapy, making high-throughput study methods in demand.
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.
