FACE™ FAK

 

Figure 1: Measurement of phosphorylated and total FAK.

NIH/3T3 cells were cultured in 96-well plates and serum-starved for 16 hours. Cells were then treated with 50 ng/ml of PDGF for 5 minutes and fixed. Total and phospho FAK were each assayed in triplicate using the phospho and total FAK antibodies included in the FACE FAK Kit. Data was plotted after correction for cell number (performed through use of Crystal Violet).

Antibody Specificities

The phospho-FAK antibody is specific for phosphorylated FAK and was raised against a synthetic phospho-peptide corresponding to residues surrounding tyrosine 397 of human FAK. This antibody recognizes FAK only when phosphorylated at tyrosine 397. It does not cross-react with related kinases. The total-FAK antibody recognizes FAK proteins regardless of the phosphorylation state.

FAK Overview

Focal adhesion kinase (FAK) is a widely expressed non-receptor cytoplasmic tyrosine kinase that is implicated in integrin-mediated signal transduction. FAK plays an important role in the control of several biological processes, including cell spreading, migration and survival. Physical interactions of FAK with the integrin cytoplasmic domain and cytoskeletal proteins talin, paxillin and/or tensin play a key role in FAK activation by facilitating its oligomerization and transphosphorylation. FAK shows a rapid increase in tyrosine phosphorylation when stimulated by diverse signaling molecules, including those that regulate embryonic development, cell proliferation, migration and apoptosis, and efficient study methods 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.

Figure 1: Flow chart of the FACE process.

Flow chart of the FACE in cell Western method that uses a cell based ELISA to measure the levels of the native and phospho forms of signaling proteins and kinases that are activated by phosphorylation.

Contents & Storage

Two (or ten) 96-well plates for culturing cells, 96 (or 5 x 96) rxns each of two primary antibodies (1 phospho-specific, 1 specific for native protein), HRP-conjugated secondary antibody, Quenching Solution, 1X Antibody Blocking Buffer, 1X Antibody Dilution Buffer, 10X PBS, 10% Triton X-100, 1% SDS Solution, Developing and Stop Solutions, and Crystal Violet Cell Quantification Solution. Storage conditions vary from room temperature to -20°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.