title: Highly sensitive detection of protein phosphorylation by using improved Phos-tag Biotin.
abstract: We have previously shown that the dinuclear zinc(II) complex Phos-tag and its derivatives act as phosphate-capture molecules in aqueous solution under conditions of neutral pH. In this study, our aim was to develop more-advanced applications for the detection of phosphopeptides and phosphoproteins by using several newly synthesized Phos-tag derivatives, including a bisbiotinylated Phos-tag (BTL-108), a tetrakisbiotinylated Phos-tag (BTL-109), and a monobiotinylated Phos-tag with a dodeca(ethylene glycol) spacer (BTL-111), as well as the commercially available product BTL-104. Among these complexes, BTL-111 showed the best performance in Western blotting by an ECL system using HRP conjugated streptavidin. In addition, in a quartz-crystal microbalance analysis of a phosphoprotein, the presence of the long hydrophilic dodeca(ethylene glycol) spacer in a novel Phos-tag sensor chip coated with BTL-111 resulted in a greater sensitivity than was achieved with a similar chip coated with BTL-104. Moreover, a peptide microarray technique using the ECL system and BTL-111 permitted high-throughput assays for the specific and highly sensitive detection of protein kinase activities in cell lysates.
authors.affiliation: Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. kinoeiji@hiroshima-u.ac.jp
authors.forename: Eiji
authors.initials: E
authors.lastname: Kinoshita
authors.forename: Emiko
authors.initials: E
authors.lastname: Kinoshita-Kikuta
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Sugiyama
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Fukada
authors.forename: Tomomitsu
authors.initials: T
authors.lastname: Ozeki
authors.forename: Tohru
authors.initials: T
authors.lastname: Koike
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry; D001710:Biotin / Q000737:chemistry*; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry; D008163:Luminescent Measurements; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D010766:Phosphorylation; D040081:Protein Array Analysis; D040901:Proteomics / Q000379:methods*; D011725:Pyridines / Q000737:chemistry*; D012680:Sensitivity and Specificity
chemical_list: C514153:1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol; D001426:Bacterial Proteins; D007202:Indicators and Reagents; D010748:Phosphopeptides; D010750:Phosphoproteins; D011725:Pyridines; D001710:Biotin; D006735:Horseradish Peroxidase