Phospho-FAK (Tyr397) STAR ELISA Kit

Phospho-FAK (Tyr397) STAR ELISA Kit

Upstate (Millipore)

FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase which localizes to focal adhesions and plays a key role in integrin-mediated signaling pathways regulating cell migration. It autophosphorylates on tyrosine 397 during adhesion and spreading, and is thought to be a critical transducer of adhesion-dependent growth and survival. FAK and it phosphorylation states have been implicated in cancer metastasis and tumor cell survival and adhesion-independent growth. Additionally, recent evidence indicates that elevation of FAK activity in human carcinoma cells is associated with increased invasive potential. A central role in tumor formation and progression suggest that FAK is an attractive target for therapeutic intervention. Cell surface integrins when engaged with their ligands lead to the recruitment of a number of intracellular proteins to specialized sites of the cytoplasmic face into focal adhesions. These focal adhesion plaques are composed of many proteins that include kinases, phosphatases, scaffolding proteins, and G-proteins. The activation of integrins leads to the tyrosine phosphorylation of several cytoplasmic proteins critical to the biochemical process, including FAK, Src, paxillin, tensin, and p130Cas. This clustering and activation of proteins at the membrane leads to the activation of many diverging signaling pathways that result in cytoskeletal re-organization. This starts with either the recruitment of Src and FAK to the plasma membrane following integrin or PI3 Kinase activation. The non-receptor protein-tyrosine kinases (PTKs) FAK and Src are key members of the focal adhesion complex. These PTKs co- Localize and associate with the transmembrane integrins and various downstream targets of certain growth factor receptors. The outcome of Src binding to FAK is the phosphorylation of several tyrosine residues in FAK and its related proteins, including the FAK autophosphorylation site Tyr397. This autophosphorylation occurs immediately after integrin clustering and allows for the association of FAK with various signaling proteins such as PI3 kinase, Grb2-Sos, p130Cas, and paxillin. The phosphorylation of FAK Tyr397 is crucial for many of the established biological roles of FAK, including cell migration, cell cycle progression, and prevention of anoikis, a form of detachment-induced apoptosis and is correlated with the invasiveness of tumors and is thought to be a pivotal player by modulating the turnover of focal adhesions to regulate cell migration. Phosphoinositide 3-Kinase (PI3 kinase or PI3K) also plays a role in cell migration via its binding to the autophosphorylation site (Tyr397) of FAK through one or both of its SH2 domains in the p85 subunit of PI3K. This is the same site in FAK that is bound by Src. In theory, the binding of PI3 kinase to FAK may be involved in activities like cell proliferation, apoptosis, and migration that are related to the phosphorylation of Tyr397. This is substantiated by the observation that AKT is downstream of PI3 kinase and is a mediator in preventing apoptosis. Therefore it is possible that FAK/PI3 kinase association may help regulate apoptosis.

Enzyme Assay

• Human
• Mouse
• Rat

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Focal Adhesion Kinase, p125FAK

FAK (Focal Adhesion Kinase) is a tyrosine kinase which localizes to focal adhesions. It autophosphorylates on tyrosine during adhesion and spreading, and is thought to be a critical transducer of adhesion-dependent growth and survival. FAK is a substrate of Src, and phosphorylation of FAK may be the mechanism by which Src-transformed cells achieve adhesion-independent growth. FAK is not known to be overexpressed in human cancer, but the activation state of FAK in cancer has not been thoroughly addressed.

Sensitivity: 1.6 Units/mL.
Range of Detection: 1.6 to 100 Units/mL

Phosphorylation

1. Multi-channel or repeating pipettes 2. Plate shaker (optional) 3. Pipettors and tips capable of accurately measuring 1-1000µL 4. Graduated serological pipettes 5. 96-well microtiter Plate Reader with 450 nm filter 6. Graphing software for plotting data or graph paper for manual plotting of data 7. Microfuge tubes for standard and sample dilutions 8. Mechanical vortex 9. 1 liter container 10. Distilled or deionized water

• 1. Capture Plate pre-coated with anti-FAK antibody: (Part No. 17-480A) One pre-coated 96-stripwell immunoplate sealed in a foil pouch.
• 2. Anti-phospho-FAK (Tyr397) detection antibody: (Part No. 17-480B) One bottle (11 mL) of anti-phospho-FAK(Tyr397) detection antibody containing sodium azide, ready to use.
• 3. ELISA Diluent: (Part No. 17-480C) One bottle (25 mL) of ELISA Diluent containing sodium azide, ready to use.
• 4. 25X ELISA Wash Buffer: (Part No. 17-480D) One bottle (50 mL) of 25X ELISA Wash Buffer.
• 5. Anti-Rabbit IgG HRP conjugate: (Part No. 17-480E) One vial (125 µL) of 100X anti-rabbit HRP conjugate containing thimerosal.
• 6. HRP Diluent: (Part No. 17-480F) One bottle (25 mL) of HRP Diluent containing thimerosal.
• 7. TMB Solution: (Part No. 17-480G) One bottle (25 mL) of stabilized tetramethylbenzidine (TMB), ready to use.
• 8. Stop Solution: (Part No. 17-480H) One bottle (25 mL) of stop solution, ready to use.
• 9. phospho-FAK (Tyr397) Standard: (Part No. 17-480I) Two vials of phospho-FAK (Tyr397) standard, lyophilized.
• 10. Plate Covers: Two plate covers.

• PTK2
• pp125FAK
• FADK
• FAK1
• FAK

FAK

Routinely evaluated by ELISA.

Chromogenic

1 year at 4°C from date of shipment