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In vitro UDP-sugar:undecaprenyl-phosphate sugar-1-phosphate transferase assay and product detection by thin layer
Kinnari B Patel1, Miguel A Valvano
1Centre for Infection and Immunity, Queen's University Belfast, Belfast, UK.
Abstract:
In vitro assays are invaluable for the biochemical characterization of UDP-sugar:undecaprenyl-phosphate sugar-1-phosphate transferases. These assays typically involve the use of a radiolabeled substrate and subsequent extraction of the product, which resides in a lipid environment. Here, we describe the preparation of bacterial membranes containing these enzymes, a standard in vitro transferase assay with solvents containing chloroform and methanol, and two methods to measure product formation: scintillation counting and thin layer chromatography.
