FAQ: Can an O-Glycoprotease reaction be scaled linearly to accommodate digestion of greater than 10 μg of glycoprotein?

Yes, the O-Glycoprotease reaction can be scaled linearly. Typical reaction conditions recommend 1 µl of O-Glycoprotease per 10 µg of substrate; reactions can be scaled linearly by maintaining an enzyme:substrate ratio of 1:10. However, the exact enzyme to substrate ratio must be determined empirically as substrate purity, tight folding, degree of glycosylation and clustering of O-glycosylation sites may affect the digestion efficiency. Longer incubation times (18 hours) and more enzyme may be necessary in some cases. It is recommended to first test the digestion efficiency at small scale (10 µg) prior to scaling up, and to monitor the efficiency of cleavage using SDS-PAGE analysis.