Competitive Inhibition Assay for CLIP-tag, SsOGT-H5, and SsOGT-MC8 Substrate Specificity

The substrate specificity of CLIP-tag, SsOGT-H⁵ and SsOGT-MC⁸ proteins were evaluated on BG-N₃ and BC-N₃ substrates by the competitive inhibition assay performed as described [15] , [27] , [29] (link). By using fixed concentrations of the fluorescent BC-TMR substrate (5 μM) and enzymes (5 μM), an increasing concentration of guanine/cytosine-azide derivatives (0–1 mM) was added to the mixtures. The reactions were incubated for 60 min at 25 °C for CLIP-tag and 65 °C for MC⁸ respectively and then stopped by adding Laemmli buffer 1 ×. Subsequently, the samples were loaded on SDS-PAGE and the fluorescent bands were measured by gel-imaging on a VersaDoc 4000™ system (Bio-Rad), by applying green LED/605 bandpass filter. Then, obtained data were plotted by IC₅₀ equation [15] , [25] . As control, 5 μM of SsOGT-H⁵ protein was incubated at 65 °C with 5 μM of the fluorescent substrate BG-FL in presence of an increasing amount of the BG/BC-azide derivatives (0–1 mM). In this case, fluorescent bands were visualized on VersaDoc 4000™ system (Bio-Rad), by applying a blue LED/530 bandpass filter.