|Product Overview:||p56Lck Kinase (Human) Assay/Inhibitor Screening Assay Kit is a single-site, non-quantitative immunoassay for kinase activity of recombinant catalytic domain of Lck. Plates are pre-coated with a newly designed "Tyrosine kinase-binding module-1", which can easily bind recombinant catalytic domain of Lck, subsequently activate Lck kinase activity on a microtiter plate. The detector antibody is PY-39, an antibody that specifically detects the phosphotyrosine residue on recombinant catalytic domain of Lck itself, which means that this kit measures the intensity of autophosphorylation of Lck catalytic domain.|
|Description:||Lck is a 56-kDa protein tyrosine kinase that is predominantly expressed in T lymphocytes. A member of the Src kinase family, it has a unique N-terminal region followed by SH3, SH2, and catalytic domains. Lck is an important protein tyrosine kinase in lymphocytes; its overexpression renders T cells hypersensitive to antigen stimulation, and an Lck-deficient T cell line, J.CaM1, exhibits dramatically reduced protein tyrosine phosphorylation following T cell receptor (TCR) cross-linking. Furthermore, genetic experiments have shown that mice deficient in Lck or expressing a dominant-negative mutant form of Lck exhibit a severe defect in T cell maturation. Lck is localized to the membrane through myristylation and palmitylation and a portion of cellular Lck associates with the cytoplasmic tail of CD4 via cysteine residues. CD4 binds to class II major histocompatibility complex molecules on antigen-presenting cells, and this interaction between CD4 and major histocompatibility complex activates Lck, perhaps through conformational changes. The Lck associated with CD4 propagates key biochemical signals in CD4 co-receptor function. Like all Src family kinases, Lck is activated and inhibited by tyrosine phosphorylation, Tyr-394 is the site of stimulatory phosphorylation, whereas Tyr-505 is the site of inhibitory phosphorylation.|
|Applications:||1) Screening inhibitors or activators of recombinant catalytic domain of Lck.2) Detecting the effects of pharmacological agents on recombinant catalytic domain of Lck.|
|Usage:||For research use only (RUO)|
|Storage:||• Upon receipt store the ATP at -20°C.• Upon receipt store all other components at 4°C; Do not expose reagents to excessive light|
|Kit Components:||Microplate: One microplate supplied ready to use, with 96 wells (12 strips of 8-wells) in a foil, zip-lock bag with a desiccant pack. Wells are coated with recombinant "Tyrosine kinase-binding module-1".10X Wash Buffer: One 100 mL bottle of 10X buffer containing 2%Tween -20.Kinase Buffer: One 20 mL bottle of 1X buffer used for Kinase Reaction Buffer and sampledilution.20X ATP: Lyophilized ATP Na2 salt. Reconstitute contents of vial with 2 mL of H2O. Mix gently until dissolved. Final concentration of ATP should be 1 mM ATP. The ATP solution can be stored in small aliquots (e.g. 100 µL) at -20°C. The 1 mM ATP stock solution must be diluted to 50 µM in Kinase Reaction Buffer at the time of the assay.HRP conjugated Detection Antibody: One bottle containing 12 mL of HRP (horseradish peroxidase) conjugated anti-phosphotyrosine monoclonal antibody (PY-39).Substrate Reagent: One bottle containing 12 mL of the chromogenic substrate, tetra-methylbenzidine (TMB). Ready to use.Stop Solution: One bottle supplied ready to use, containing 12 mL of 1.25 N H2SO4. Ready to use.|
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