HTRF Human & Mouse Phospho-FAK (Tyr397) Detection Kit, 10,000 Assay Points

Phospho-FAK (Tyr397) assay principle

The Phospho-FAK (Tyr397) assay measures FAK (Focal Adhesion Kinase) when phosphorylated at Tyr397. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-FAK (Tyr397) assay uses 2 labeled antibodies, one with a donor fluorophore, the other with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Phospho-FAK (Tyr397) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Phospho-FAK (Tyr397) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-FAK (Tyr397) one-plate assay protocol

Detection of Phosphorylated FAK (Tyr397) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

HTRF phospho and Total FAK assays to monitor pharmacology of FAK inhibitors

HUVEC human endothelial cells were plated in a 24-well collagen I plate (400,000 cells/well) in complete culture medium and incubated at 37°C, 5% CO2. The day after, cells were incubated in serum-free conditions. The following day, cells were treated with increasing concentrations of VEGF for 5 min in serum-free medium. After medium removal, cells were then lysed with 250 µL supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the premixed HTRF phospho-FAK (Tyr397) or Total-FAK detection reagents. The HTRF signal was recorded after an overnight incubation.

HUVEC cells stimulated with VEGF showed a dose dependent increase in FAK phosphorylation on Tyrosine 397. VEGF regulates FAK phosphorylation and plays a role in angiogenesis and cell growth.

HTRF phospho-FAK cellular assays compared to Western Blot

The mouse NIH/3T3 cell line was seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2 until 90% confluency was reached. Cells were then lysed with 3 mL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10-minute centrifugation.

Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF® phospho-FAK detection reagents. Equal amounts of lysates were used to establish a side-by-side comparison between HTRF and Western Blot.

FAK simplified pathway

This multi-domain protein changes conformation upon activation by cell surface receptors, mainly by integrins, cytokine receptors (IL1βR, TNFαR), growth factor receptors (EGFR, VEGFR, PDGFR), and GPCRs (LPA, bombesin). The cytoplasmic inactive FAK monomer is recruited to the plasma membrane by activated receptors that yield to a transient dimerization and subsequent activation by autophosphorylation on tyrosine 397. This is a docking site for Src family kinases, allowing phosphorylation of other residues. Fully activated FAK displays binding sites for FAK substrates including Grb2, PI3K, and cadherins. FAK serves as a central regulator of ECM/integrin signaling, is required in the regulation of assembly/disassembly of focal adhesions, and plays a role in cell adhesion, migration, survival, and mechanosensing. In cadherin signaling, the regulation of adherens junctions between endothelial/epithelial cells enables FAK to play a role in cell junction, vascular permeability, and angiogenesis. FAK can activate various substrates and regulate multiple signaling pathways, such as Rho GTPases/actin (cytoskeleton reorganization/cell migration), Cadherin/β-catenin (cell junctional stability/vascular permeability), PI3K/AKT (cell survival), MAPKs ERK & JNK (cell proliferation), and p53/MDM2 (anti-apoptosis).