TABLE 1.

OGT and OGA activity assays.

Assay	Substrate and measurement	Application
OGT activity	UDP-[3H]GlcNAc; CKII aa340-352; measure µCi GlcNAc incorporated (Zachara et al., 2011)	Can be performed with crude preparations (∼20–50 μg protein) or purified OGT enzyme (∼0.2–1.0 μg protein), sensitive to salt inhibition (Zachara et al., 2011; Groves et al., 2017)
OGT activity	Measure chemosensor binding, since it binds stronger with UDP than UDP-sugar (Kim, 2011)	The chemosensor binds more strongly to UDP than to the UDP-GlcNAc nucleotide-sugar donor and may be used to detect changes in O-GlcNAc incorporation. The chemosensor used may have nonspecific interactions with other cellular components
OGT activity	Measure ligand displacement using fluorescent UDP-GlcNAc analogs and an active sOGT enzyme (Kim, 2011)	Purified enzyme is needed. This method can be used for the screening of OGT inhibitors
OGA activity	Measure absorbance or fluorescence changes for synthetic substrates pNP-β-GlcNAc (400 nm) (Zachara et al., 2011) or 4MU-β-GlcNAc (excitation 360 nm, emission 450 nm) (Groves et al., 2017)	Can be performed with cell extracts (20–50 μg protein); GalNAc can be included in the reaction to inhibit lysosomal hexosaminidases A and B at a concentration that does not inhibit OGA, or the activities on GalNAc substrates can be subtracted from those on GlcNAc substrates (Zachara et al., 2011; Groves et al., 2017)