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首页> 外文期刊>Electrophoresis: The Official Journal of the International Electrophoresis Society >A CE-based assay for human protein kinase CK2 activity measurement and inhibitor screening.
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A CE-based assay for human protein kinase CK2 activity measurement and inhibitor screening.

机译:基于CE的人体蛋白激酶CK2活性测定和抑制剂筛选方法。

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摘要

A new assay for protein kinase CK2 activity determination based on the quantification of a phosphorylated substrate was developed. The common CK2 substrate peptide RRRDDDSDDD, conjugated with the fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid at the C-terminus served as the analyte. By means of CZE using 2 mol/L acetic acid as electrolyte and UV detection at 214 nm, the non-phosphorylated and the phosphorylated peptide variants could be resolved within 6 min from a complex assay mixture. By this means, activity of human CK2 could be monitored by a kinetic, as well as an endpoint, method. Inhibition of human recombinant CK2 holoenzyme by 6-methyl-1,3,8-trihydroxyanthraquinone and 4,5,6,7-tetrabromobenzotriazole resulted in IC(50) values of 1.33 and 0.27 microM, respectively, which were similar to those obtained with the standard radiometric assay. These results suggest that the CE/UV strategy described here is a straightforward assay for CK2 inhibitor testing.
机译:建立了一种基于磷酸化底物定量的蛋白激酶CK2活性测定的新方法。在C-末端与荧光团5-[(2-氨基乙基)氨基]萘-1-磺酸缀合的普通CK2底物肽RRRDDDSDDD用作分析物。通过使用2 mol / L乙酸作为电解质的CZE和214 nm处的UV检测,可以从复杂的测定混合物中在6分钟内分辨出未磷酸化和磷酸化的肽变体。通过这种方式,可以通过动力学方法以及终点方法来监测人CK2的活性。 6-甲基-1,3,8-三羟基蒽醌和4,5,6,7-四溴苯并三唑对人重组CK2全酶的抑制作用导致IC(50)值分别为1.33和0.27 microM,这与使用标准辐射测定。这些结果表明,此处描述的CE / UV策略是用于CK2抑制剂测试的直接方法。

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