Endo H/Endo Hf Protocol

Typical reaction conditions are as follows:

  • Combine 1-20 μg of glycoprotein, 1 μl of 10X Glycoprotein Denaturing Buffer and H20 (if necessary) to make a 10 μl total reaction volume.
  • Denature glycoprotein by heating raection at 100°C for 10 minutes.
  • Make a total reaction volume of 20 μl by adding 2 μl of 10X GlycoBuffer 3, H20 and 1-5 μl Endo H.
  • Incubate reaction at 37°C for 1 hour.

Notes
  • Reactions may be scaled-up linearly to accommodate larger reaction volumes.
  • Enzymatic activity is not affected by SDS
  • To deglycosylate a native glycoprotein, longer incubation timeas well as more enzyme may be required.
  • Activity at different temperatures was measured after a 1 hour incubation of a glycosidase and denatured RNase B at the given temperatures:
    37°C - 100%
    30°C - 65%
    25°C - 40%
    17°C - 25%
    2°C - 0 %