GDP-Glo Glycosyltransferase Assay
The GDP-Glo Glycosyltransferase Assay is a bioluminescent assay for detecting the activity of glycosyltransferases that use GDP-sugars as donor substrates and release GDP as a product. Glycosylating reactions catalyzed by glycosyltransferases (GTs) are central to many biological processes, including cell-cell interactions, cell signaling and bacterial cell wall biosynthesis. Because of the importance of this class of enzymes, there is a need for biochemical assays to monitor their activity, their mode of regulation, and to search for their selective and potent inhibitors. Glycosyltransferases transfer sugar from a nucleotide-glycosyl donor to an acceptor molecule. GDP-sugar is one of the most utilized sugar donors for glycosylating enzymes after UDP-sugars (e.g., GDP-fucose and GDP-mannose). In a glycosyltransferase reaction, the GDP moiety is released as a product. Therefore, an assay that detects GDP as product of these reactions would be suitable for monitoring the activity of all the GDP-sugar-utilizing glycosyltransferases.The GDP-Gloª Glycosyltransferase Assay is a homogeneous, single-reagent-addition method to rapidly detect GDP formation in glycosyltransferase reactions. After the glycosyltransferase reaction, an equal volume of GDP Detection Reagent is added to simultaneously convert the GDP product to ATP and generate light in a luciferase reaction. The light generated is detected using a luminometer, and the light output is proportional to the concentration of GDP from low nM to 25microM. Luminescence can be correlated to GDP concentration by using a GDP standard curve. The assay is easy to use and highly sensitive, two features that are desirable and essential for measuring the activity of different GDP-sugar-utilizing glycosyltransferases such as fucosyltransferases. Therefore, the GDP detection assay uses less enzyme in glycosyltransferase reactions. This assay is fast and simple. The GDP-Glo Assay is performed in a single well of a multiwell plate and can be used to detect glycosyltransferase activity in as little as a 5microl reaction.