کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10533823 | 961894 | 2005 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Fluorometric TLC assay for evaluation of protein kinase inhibitors
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
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چکیده انگلیسی
A fluorometric assay for measuring protein kinase activity has been developed. The assay is based on the separation of fluorescently marked substrate 5-carboxytetramethylrhodamine-kemptide (5-TAMRA-kemptide) from its phosphorylated counterpart by TLC and quantification of the product ratiometrically by fluorescence imaging. The utility of the assay was demonstrated by measuring the activity of cAMP-dependent protein kinase. 5-TAMRA-kemptide was characterized as a substrate of this kinase by the kinetic parameters Kmapp and Vmax. The attachment of 5-TAMRA dye to the N terminal of kemptide decreased the Kmapp value but did not have a significant effect on the rate and stoichiometry of the phosphorylation reaction. The inhibitory potency of three known inhibitors was evaluated with the new assay. The closeness of the obtained inhibitory activities of the compounds to the activities determined with the phosphocellulose paper-binding assay, as well as the Zâ² factor value of 0.5, demonstrates the reliability of the new assay for evaluation of inhibitors of protein kinases.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 340, Issue 1, 1 May 2005, Pages 165-170
Journal: Analytical Biochemistry - Volume 340, Issue 1, 1 May 2005, Pages 165-170
نویسندگان
Kaido Viht, Angela Vaasa, Gerda Raidaru, Erki Enkvist, Asko Uri,