Protocol for quantification of UDP-GlcNAc using an enzymatic microplate assay

Abstract

Summary: Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is the end product of the hexosamine biosynthetic pathway and the substrate for protein O-linked N-acetylglucosaminylation (O-GlcNAcylation). Here, we present a protocol for the quantification of UDP-GlcNAc using an enzymatic microplate assay. We also detail procedures for the extraction of polar metabolites and total protein fraction for the parallel quantification of UDP-GlcNAc and the western blot analysis of O-GlcNAcylated proteins, O-linked N-acetylglucosamine transferase, and O-GlcNAcase from the same sample.For complete details on the use and execution of this protocol, please refer to Sunden et al. (2023).1 In addition, a preview article by Chatham et al. provides a useful summary of the method.2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Metabolism
• Molecular Biology