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Updated: Nov 11, 2025
A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells
Published on: April 11, 2014
Rapid identification of magnesium ascorbyl phosphate utilizing phosphatase through a chromogenic change-coupled
Yuli Wang1, Wenyue Hu1, Zixin Deng1
1State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People's Republic of China.
Abstract:
In this study, we report a chromogenic reaction between magnesium ascorbyl phosphate (MAP) and ferric chloride to generate a Brown-Red clathrate, while the Treated MAP by phosphatases forms Colorless (BRTC) product with ferric chloride. The BRTC was indicative of phosphatase activity-mediated excision of phosphorous group from MAP and utilized to screen phosphatases from bacterial cell lysates. From ten tested strains, BRTC was observed in the cell lysate of Salmonella enterica subsp. enterica serovar Cerro 87. BRTC was again employed to track phosphatase activity of the resuspensions of the ammonium sulfate graded precipitations of the cell lysate. Two phosphatases, PhoN and YcdX, were identified by LC-MS/MS analysis in the protein fraction giving most obvious BRTC phenotype and validated by examination of in vitro activity of the purified proteins. KEY POINTS: • BRTC is labelling-free, naked-eye visible, and independent of any facilities. • BRTC can directly screen phosphatases from microbial cell lysates. • Using BRTC system, two phosphatases were identified in Salmonella enterica subsp. enterica serovar Cerro 87.
