YOUR CART
The transcription factor NFκB (nuclear factor κB) is a key component for the inducible expression of a wide variety of cellular and viral genes. NFκB is composed of a heterodimer of p65 and p50 subunits in most cell types and is sequestered in the cytoplasm by its inhibitory proteins, the IκBs. During the phosphorylation and degradation of IκBs, NFκB p65 is phosphorylated on multiple resides, each triggered by different stimuli but essential for maintaining NFκB transcriptional activation. Because of its role in regulating inflammatory and immune responses, high-throughput study methods for monitoring NFκB 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.
