Endo F2 Reaction Protocol (P0772)
Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Typical reaction conditions are as follows:
- Combine 20 µg of glycoprotein, 1 µl of GlycoBuffer 4 (10X) and H2O (if necessary) to make a 10 µl total reaction volume.
- Add 1 µl Endo F2
- Incubate reaction at 37°C for 1 hour.
- The reaction may be scaled-up linearly to accommodate large amounts of glycoprotein and larger reaction volumes.
- The optimal pH for the cleavage of biantennary (i.e. porcine fibrinogen) glycans is pH 4.5 [GlycoBuffer 4 (10X)]. However, cleavage can also be achieved at pH 6.0 [GlycoBuffer 3 (10X)].
- Endo F2 is typically used under non-denaturing (native) conditions, as denaturation is not necessary for optimal activity of the enzyme. However, Endo F2 is active in denaturing conditions. Concentrations of 0.5% SDS do not inhibit Endo F2 activity when used in the presence of 1% NP-40.