Takiyama, K., Kinoshita, E., Kinoshita-Kikuta, E., Fujioka, Y., Kubo, Y. and Koike T. (2009) A Phos-Tag-Based Fluorescence Resonance Energy Transfer System for the Analysis of the Dephosphorylation of Phosphopeptides. Analytical Biochemistry, 388, 235-241.
http://dx.doi.org/10.1016/j.ab.2009.02.039

has been cited by the following article:

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

  AUTHORS: Emiko Kinoshita-Kikuta, Hiromasa Kurosaki, Natsumi Kunisada, Eiji Kinoshita, Tohru Koike

  KEYWORDS: Fluorescence Resonance Energy Transfer, Enzyme Assays, Enzyme Inhibitors, Phos-Tag, Alkaline Phosphatase

  JOURNAL NAME: American Journal of Analytical Chemistry, Vol.5 No.12, August 29, 2014

  ABSTRACT: 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.