• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>American Journal of Analytical Chemistry >A Phos-Tag-Based Fluorescence Quenching System for Activity Assay and Inhibitor Screening for Alkaline Phosphatase
【24h】

A Phos-Tag-Based Fluorescence Quenching System for Activity Assay and Inhibitor Screening for Alkaline Phosphatase

机译：基于磷酸标记的荧光猝灭系统，用于碱性磷酸酶的活性测定和抑制剂筛选

获取原文
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here we introduce a novel FRET-based fluorescence quenching system for assaying the activity of alkaline phosphatase (AP) by using a phos-phate-binding tag molecule, Phos-tag {1,3-bis[bis(pyridine-2-ylmethyl)amino]propan-2-olato dizinc(II) complex}, attached to a nonfluorescent 4-{[4-(dimethylamino)phenyl]diazenyl}benzoyl (Dabcyl: λmax 475 nm) dye group. The fluorogenic biomolecule riboflavin 5’-phosphate (FMN: λem 525 nm) was used as an AP substrate. The Dabcyl-labeled Phos-tag specifically captured FMN to form a stable 1:1 complex, resulting in efficient fluorescence quenching. The quenching efficiency was more than 95% for a mixture of 12 μM FMN and 13.5 μM Dabcyl-labeled Phos-tag in aqueous solution at pH 7.4 and 25°C. When FMN was dephosphorylated with AP, riboflavin was released into the solution and fluorescence from the flavin moiety appeared. By using this quenching system, we succeeded in detecting time- and dose-dependent dephosphorylation of FMN by AP under near-physiological conditions.
机译：荧光共振能量转移（FRET）是两个染料分子的电子激发态之间的距离依赖相互作用。在这里，我们介绍了一种新型的基于FRET的荧光猝灭系统，该系统通过使用磷酸酯结合标签分子Phos-tag {1,3-bis [bis（pyridine-2-ylmethyl）氨基]丙烷-2-olato二锌（II）络合物}，连接至非荧光的4-{[4-（二甲基氨基）苯基]二氮烯基}苯甲酰基（Dabcyl：λmax475 nm）染料基团。荧光生物分子核黄素5'-磷酸盐（FMN：λem525 nm）用作AP底物。 Dabcyl标记的Phos-tag特异性捕获FMN以形成稳定的1：1络合物，从而实现有效的荧光猝灭。在pH 7.4和25°C的水溶液中，12μMFMN和13.5μMDabcyl标记的Phos-tag的混合物的猝灭效率超过95％。当FMN用AP进行去磷酸化时，核黄素释放到溶液中，并出现黄素部分的荧光。通过使用该淬灭系统，我们成功地在近生理条件下检测了AP对FMN的时间和剂量依赖性脱磷酸作用。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号