Doyle Liam, Ovchinnikova Olga G, Whitfield Chris
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
Methods Mol Biol. 2019;1954:151-159. doi: 10.1007/978-1-4939-9154-9_12.
The incorporation of fluorescent tags into synthetic acceptor molecules for in vitro biochemical assays allows quick and easy detection of enzyme activity. Reaction products can be separated via thin-layer chromatography and visualized under UV light for rapid detection of reaction progress. Subsequent structural analysis of these reaction products through the use of NMR spectroscopy and mass spectrometry allows for complete functional characterization of enzyme activity. Here we describe an application of this technique which was previously used to functionally characterize a dual-domain glycosyltransferase enzyme, KpsC, involved in capsular polysaccharide biosynthesis in Escherichia coli.
