Invitrogen™ Human FAK (Phospho) [pY397] ELISA Kit

Description

ELISA

The Human FAK (Phospho) [pY397] ELISA Kit is designed to detect and quantify the level of FAK (Phospho) [pY397]) in fresh or frozen human and mouse cell lysates. The assay recognizes both natural and recombinant FAK (Phospho) [pY397]. Principle of the method A monoclonal capture antibody specific for FAK (Phospho) [pY397] has been coated onto the wells of the 96-well plate. During the first incubation, standards of known content and unknown samples are pipetted into the wells and the antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for the target protein is added to the wells and serves as a detection antibody by binding to the immobilized protein captured during the first incubation. After washing, a horseradish peroxidase labeled anti-rabbit IgG is added. This binds to the detection antibody to complete the four member sandwich. After a third incubation and washing to remove all the unbound enzyme, a substrate solution (TMB) is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of target protein present in the original specimen and the optical density can be read on a standard microplate reader. Rigorous validation Each manufactured lot of this ELISA kit is quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency. See manual for more information on validation.

Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor protein tyrosine kinase that acts as a substrate for Src and is a key element of integrin signaling. FAK plays an important role in cell spreading, differentiation, migration, cell death, and acceleration of the G1 to S phase transition of the cell cycle. FAK has a central catalytic domain and a C-terminal tail that localizes it to focal adhesions, which are sites where cells attach to the extracellular matrix via surface integrin receptors. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Adhesion of murine NIH3T3 fibroblasts to fibronectin promotes association of the Grb2 adapter protein and c-Src PTK with FAK in vivo, and also results in activation of the ERK2 MAP kinase. In v-Src-transformed NIH3T3, the association of v-Src, Grb2, and Sos with FAK is independent of cell adhesion to fibronectin. In vitro the Grb2 SH2 domain binds directly to tyrosine-phosphorylated FAK, and the binding site has been identified as Tyr925 by site directed mutagenesis. Several transcript variants encoding different isoforms have been found for the FAK gene, but the full-length natures of only three of them have been determined.

Specifications

• Accession Number: P34152,Q05397
• Assay Range: 1.6 to 100 U/mL
• Assay Sensitivity: <0.9 U/mL
• Concentration: 1.6 U/mL
• Conjugate: HRP
• Product Type: ELISA
• Sample Type: Cell Lysates
• Substrate Type: Cytoplasmic Tyrosine Kinases
• Target Species: Mouse, Rat
• For Use With (Equipment): Colorimetric Microplate Reader
• Gene ID (Entrez): 14083, 5747
• Gene Symbol: Ptk2
• Incubation Time: 4 hr.
• Interassay CV: 8%
• Intraassay CV: 4
• Kit Contents: Pre-coated 96 well plate, Standard, Standard Dilution Buffer, Detection Antibody, anti-rabbit-HRP, HRP Diluent, Wash Buffer, Chromogen, Stop Solution, Adhesive Plate Covers
• Label or Dye: HRP
• Marker Gene: FAK
• Quantity: 96 Tests
• Regulatory Status: RUO
• Sample Volume: Cell Lysate 10 μL
• Storage Requirements: 2°C to 8°C
• Target: FAK
• Test Time: 1 hrs 20 mins
• Total Assay Time: 4 hrs