The phospho-FAK (Tyr397) kit enables the cell-based quantitative detection of phosphorylated FAK as a readout of integrin engagement by ECM, growth factor stimulation, and cell migration.

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  • High sensitivity High sensitivity
  • Ready-to-use Ready-to-use
  • No-wash No-wash
  • Low sample consumption Low sample consumption

The phospho-FAK (Tyr397) kit enables the cell-based quantitative detection of phosphorylated FAK as a readout of integrin engagement by ECM, growth factor stimulation, and cell migration.

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Overview

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.

Benefits

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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-y397-assay-principle

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-y397-2-plate-assay-protocol

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.

phospho-fak-y397-1-plate-assay-protocol

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 HTRF Phospho & Total FAK assays on the MCF7 cells treated with pervanadate
Validation of the HTRF Phospho & Total FAK assays on HeLa cells treated with pervanadate

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 HTRF Phospho & Total FAK assays on the HeLa cells treated with PF562271
Validation of the HTRF Phospho & Total FAK assays on NIH3T3 cells treated with PF562271

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.

Validation of the HTRF Phospho & Total FAK assays on the HUVEC cells treated with VEGF

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.

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

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).

Pathway FAK Phospho
HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

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A solution for phospho-protein analysis in metabolic disorders - Posters

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

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HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

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Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Investigating kinase activity in a cellular context

HTRF cellular assays - Scientific Presentations

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Save time and money

Switch to HTRF assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Inflammation cell by cell

HTRF solutions for each cell type - Flyers

Product Insert FAK P-Y397 Kit / 64FAKPEG-64FAKPEH

64FAKPEG-64FAKPEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

A collection of the most important NAFLD pathways

Useful overview of today’s NAFLD knowledge - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

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