YOUR CART
Glycogen synthase kinase-3 (GSK3), originally discovered as a serine/threonine protein kinase that phosphorylates and inactivates glycogen synthase, is now known to regulate a diverse array of cellular processes. Inappropriate regulation of GSK3 signaling is thought to play a role in the development of type II diabetes mellitus, neuronal cell loss in Alzheimer’s disease and cancer. The two major isoforms of GSK3 in mammalian tissues (GSK3α and 3β) are structurally similar but not functionally equivalent. GSK3β activity is relatively high in resting cells, and it can be positively or negatively regulated by various stimuli. GSK3β is a promising drug target and high-throughput study methods are 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.
