The required amount of Endo-β-Galactosidase depends on the specific substrate. As an initial guideline, use 1–2 μl of enzyme for 10-20 μg of glycoprotein in a 10 μl reaction for one hour at 37°C.
In our studies, with a sugar-conjugated protein, we observed that the enzyme functions effectively across a broad range of protein-to-enzyme molar ratios, from 2:1 to 150:1, which translates to a roughly 50- to 3500-fold sugar moiety-to-enzyme ratio under optimal conditions. We recommend beginning with a 10:1 to 20:1 ratio and conducting a titration experiment to optimize conditions for your specific substrate.
