Revvity, 64FAKPEH, HTRF Human and Mouse Phospho-FAK (Tyr397) Detection Kit, 10,000 Assay Points

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.
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) 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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 2 Lysis Buffer 3
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Therapeutic Area-Cardiovascular NASH/Fibrosis Oncology & Inflammation
Unit Size-10,000 assay points