Protocol for Endo H/Hf Non-Denaturing P0702 and P0703

Non-Denaturing Reaction Conditions:

When deglycosylating a native glycoprotein it is recommended that an aliquot of the glycoprotein is subjected to the denaturing protocol to provide a positive control for the fully deglycosylated protein. The non-denatured reaction can then be compared to the denatured reaction to determine the extent of reaction completion.

  1. Combine 20 µg of glycoprotein, 2 µl of GlycoBuffer 3 (10X) and H2O (if necessary) to make a 20 µl total reaction volume.
  2. Add 2-5 µl Endo H, mix gently.Incubate reaction at 37°C for 4 - 24 hours.
    Note: To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required. For more complete deglycosylation  an overnight digestion is recommended.
  3. Analyze by method of choice. The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.