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.
- Combine 20 µg of glycoprotein, 2 µl of GlycoBuffer 3 (10X) and H2O (if necessary) to make a 20 µl total reaction volume.
- 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.
- Analyze by method of choice. The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.