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Protein Modification

FACE™ c-Jun

in-cell Western analysis for phospho c-Jun (S63 or S73)

 

c-Jun is a component of the transcription factor AP-1, which binds and activates transcription at TRE/AP-1 elements. The c-Jun oncogene encodes a nuclear protein, p39, that interacts with the AP-1 c-Fos oncogene product and forms a transacting heterodimer. c-Jun plays an important role in the regulation of gene expression and signal transduction processes and is also suggested to be actively involved in the differentiation of B-chronic lymphocytic leukemia cells and Hodgkin’s Reed-Sternberg cells as well as in the malignant transformation of melanocytes, which makes it a promising drug target.

 

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.

 
FACE flow chart
 
Figure 1: Flow chart of the FACE process.