YOUR CART
Bad (Bcl-2 antagonist of cell death) is a proapoptotic member of the Bcl-2 family that displaces Bax from binding to Bcl-2 and Bcl-xL, which results in cell death. Bad forms heterodimers with other Bcl-2 family members and localizes to the outer mitochondrial membrane. When phosphorylated in the presence of survival signals, Bad translocates to the cytoplasm and blocks the induction of apoptosis. In the absence of survival signals, dephosphorylated Bad is localized in the outer membrane of the mitochondria where it activates mitochondrial-mediated apoptotic pathways. Thus, Bad is a unique Bcl-2 family member that plays a key role in the transduction of upstream survival signals to the Bcl-2 apoptosis decision machinery, and high-throughput study methods for monitoring Bad phosphorylation are in high 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.
