An enzymatic colorimetric assay for glucose-6-phosphate |
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| Authors: | Zhu Aiping Romero Roberto Petty Howard R |
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| Institution: | aDepartment of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, MI 48105, USA;bPerinatology Research Branch, Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) of National Institutes of Health, Bethesda, MD, and Detroit, MI 48201, USA;cCenter of Molecular Medicine and Genetics, Wayne State University, Detroit, MI, and Hutzel Women’s Hospital at the Detroit Medical Center, Detroit, MI 48201, USA;dDepartment of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48105, USA |
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| Abstract: | A specific colorimetric assay for the determination of glucose-6-phosphate (G6P) was developed. This assay is based on the oxidation of G6P in the presence of glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP+); the NADPH thereby generated reduces the tetrazolium salt WST-1 2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt] to water-soluble yellow-colored formazan with 1-methoxy-5-methylphenazium methylsulfate (1-mPMS) as an electron carrier. The assay is optimized for reaction buffer pH, enzyme/dye concentration, and reaction time course. The limit of detection of the assay is 0.15 μM (15 pmol/well). The usefulness of the assay is demonstrated by the accurate measurement of the G6P concentration in fetal bovine serum (FBS). |
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| Keywords: | WST-1 Glucose-6-phosphate Colorimetric assay Glucose-6-phosphate dehydrogenase |
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