title: A new fluorescent quenching method for the determination of phosphoproteins by using calconcarboxylic acid.
abstract: A fluorescent quenching detection method for phosphoproteins in SDS-PAGE by using calconcarboxylic acid (CCA) was described. In this method, the fluorescence intensity of CCA was greatly increased with the presence of Al(3+) in the gel background, while in zones where phosphoproteins are located this intensity was absent because of fluorescence quenching phenomenon through the formation of CCA-Al(3+) -phosphoprotein appended complex. Approximately 4-8 ng of phosphoproteins can be selectively detected within 1 h (1D SDS-PAGE), which is similar to that of the most commonly used Pro-Q Diamond stain. The specificity of this novel technique for phosphoproteins was confirmed by dephosphorylation, Western blot, and LC-MS/MS analysis, respectively. Furthermore, to better understand the newly developed method, the detection mechanism of CCA stain was explored by fluorescent spectrometry. According to the results, it is believed that CCA stain may provide a new choice for selective, economical, MS compatible, and convenient visualization of gel-separated phosphoproteins.
authors.affiliation: Zhejiang Provincial Key Laboratory of Biopharmaceuticals, Wenzhou Medical University, Wenzhou, Zhejiang, P. R. China; Wenzhou Undersun Biotechnology Co., Ltd, Wenzhou, Zhejiang, P. R. China.
authors.forename: Zhongxin
authors.initials: Z
authors.lastname: Zhu
authors.forename: Xinliang
authors.initials: X
authors.lastname: Zhu
authors.forename: Jiayi
authors.initials: J
authors.lastname: Shen
authors.forename: Ayi
authors.initials: A
authors.lastname: Zhou
authors.forename: Maowei
authors.initials: M
authors.lastname: Ni
authors.forename: Litai
authors.initials: L
authors.lastname: Jin
authors.forename: Weitao
authors.initials: W
authors.lastname: Cong
mesh_terms: D000595:Amino Acid Sequence; D001391:Azo Compounds / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008969:Molecular Sequence Data; D009284:Naphthols / Q000737:chemistry*; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D012680:Sensitivity and Specificity
chemical_list: D001391:Azo Compounds; D005456:Fluorescent Dyes; D009284:Naphthols; D010750:Phosphoproteins; C084567:calconcarboxylic acid
keywords: Calconcarboxylic acid; Fluorescent quenching; Phosphoprotein detection; Pro-Q Diamond