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  • 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 G5 Reaction Buffer, 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 %