A microtiter-based assay for the detection of protein tyrosine kinase activity.
نویسندگان
چکیده
A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [32P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation.
منابع مشابه
Deficient Expression of Bruton's Tyrosine Kinase in Monocytes from X-Linked Agammaglobulinemia as Evaluated by a Flow Cytometric Analysis and its Clinical Application to Carrier Detection
Background: The B-cell defect in X-linked agammaglobulinemia (XLA) is caused by mutations in the gene for Bruton's tyrosine kinase (BTK). BTK mutations result in deficient expression of BTK protein in peripheral blood monocytes. Methods: Using the anti-BTK monoclonal antibody (48-2H), a flow cytometric analysis of intra cytoplasmic BTK protein expression in monocytes was performed to identify I...
متن کاملSFHS: Reusable catalyst for the synthesis of polyhydroquinoline derivatives and its molecular docking studies against tyrosine protein kinase
An efficient synthesis of polyhydroquinoline derivatives is achieved via Hantzsch condensation reaction between aldehydes, dimedone, ethyl acetoacetate and ammonium acetate in the presence of catalytic amount of SFHS in ethanol. This method gives remarkable advantages such as shorter reaction time, simple workup procedure and good to excellent yields. Furthermore the catalyst can be recovered c...
متن کاملSFHS: Reusable catalyst for the synthesis of polyhydroquinoline derivatives and its molecular docking studies against tyrosine protein kinase
An efficient synthesis of polyhydroquinoline derivatives is achieved via Hantzsch condensation reaction between aldehydes, dimedone, ethyl acetoacetate and ammonium acetate in the presence of catalytic amount of SFHS in ethanol. This method gives remarkable advantages such as shorter reaction time, simple workup procedure and good to excellent yields. Furthermore the catalyst can be recovered c...
متن کاملPhosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation
Background: When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kina...
متن کاملReceptor Tyrosine Kinase Inhibitory Activities and Molecular Docking Studies of Some Pyrrolo[2,3-d]pyrimidine Derivatives
In this study, we aimed to determine VEGFR-2, EGFR and PDGFR-β tyrosine kinase inhibitory activities of some pyrrolo[2,3-d]pyrimidine derivatives previously synthesized and showed potent cytotoxic and apoptotic effects against several cancer cell lines by our group and to evaluate the relationships between inhibitory activities and binding properties of the active compounds by molecular docking...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Analytical biochemistry
دوره 190 2 شماره
صفحات -
تاریخ انتشار 1990