Phospho-FAK (Tyr397) Cellular Kit

This HTRF cell-based assay conveniently and accurately quantifies phosphorylated FAK at Tyr397 (residue for human, rat, and chicken, equivalent to Y428 for mouse). FAK, or Focal Adhesion Kinase, is a cytoplasmic tyrosine kinase controlling cell adhesion, migration, proliferation, and survival. FAK is a central regulator of ECM/integrin and cadherin signaling, and plays a role in cell adhesion, cell junction, migration, survival, and mechanosensing. FAK can be activated by Growth Factor Receptors, Cytokine Receptors, and GPCRs. Increased FAK expression and phosphorylation is associated with increased tumor cell adhesion, migration, invasion in several cancers, and with fibroblast migration on fibronectin in tissue fibrosis.

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How it works

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)assay principle

Phospho-FAK (Tyr397) 2-plate assay protocol

The 2-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)2-plate assay protocol

Phospho-FAK (Tyr397) 1-plate assay protocol

Phospho-FAK (Tyr397) 1-plate assay protocol

Assay Validation

1. Accumulation of phosphorylated FAK (Tyr397) in human cancer cell lines

Human MCF7 or HeLa cells were plated in a 96-well plate (100,000 cells/well) in complete culture medium and incubated at 37°C, 5% CO2. The day after, cells were treated for 10 min in the presence or absence of Pervanadate. After medium removal, cells were then lysed with 50 µL of 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. Treatment with pervanadate, a phosphatase inhibitor, lead to a significant increase in the phosphorylation of FAK on Tyrosine 397. The total amount of FAK protein remained unaffected by the pervanadate treatment.

Validation of the Cisbio HTRF Phospho & Total FAK assay on the human MCF7 cell line treated with pervanadate

Validation of the Cisbio HTRF Phospho & Total FAK assay on the human THP1 cell line treated with pervanadate

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

NIH/3T3 mouse fibroblasts or human HeLa cells were plated in a 96-well plate (100,000 cells/well) in complete culture medium and incubated at 37°C, 5% CO2. The day after, cells were treated with increasing concentrations of FAK inhibitors for 30 min. After medium removal, cells were then lysed with 50 µL of 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.

Treatment with PF-562271 lead to a dose dependent decrease in FAK phosphorylation on Tyrosine 397 in both Human HeLa and Mouse NIH/3T3 cell lines. The total amount of FAK protein remained unaffected by the pervanadate treatment.

Validation of the Cisbio HTRF phospho and total FAK on human HeLa cells treated with PF562271 FAK inhibitor

Validation of the Cisbio HTRF phospho and total FAK on mouse NIH3T3 cells treated with PF562271 FAK inhibitor

3. HTRF FAK assays as readouts to monitor growth factor receptors activation

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.

4. HTRF phospho-FAK cellular assays compared to Western Blot

Comparison between WB and HTRF Phospho FAK assay on NIH/3T3 cells

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.

Using the HTRF® Phospho-FAK kit, just 5,000 cells/well were sufficient to detect a significant signal while 10,000 cells were needed for minimal ECL signal detection using Western Blot. The HTRF assay is therefore twice as sensitive as the Western Blot technique.

Simplified pathway

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).

FAK signaling pathway

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
FAK phospho-Y397 kit - 500 tests64FAKPEG
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FAK phospho-Y397 kit - 10,000 tests64FAKPEH
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FAK phospho-Y397 kit - 50,000 tests64FAKPEY
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Companion products

DescriptionCat. noProduct insertMSDS
FAK phospho-Y397 kit - control lysate64FAKTDA
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