MGAT5 Activity Assay

Briefly, MGAT5 activity was assayed in the separate tube in the absence of MnCl₂. After incubation at 37°C for 5 hours, the reactions were stopped by heating at 100°C for 3 mins and the samples were centrifuged at 5000 rpm in an Eppendorf tube for 15 min. An aliquot (20 μl) of each sample was applied to ODS C₁₈ column to separate the products of MGAT5 from the acceptor substrate by High Performance Liquid Chromatography (HPLC) method. All the samples were assayed in duplicate. The enzyme activities were calculated according to the peak areas of the products and expressed as GlcNAc (pmol) transferred/h/mg protein.