MilliporeSigma
HomeProtein Labeling & ModificationN-Glycosidase F Protocol & Troubleshooting

N-Glycosidase F Protocol & Troubleshooting

Protocol

Using N-glycosidase F (Product No. NGLYF-RO) with less soluble proteins in PBS and other non-denaturing buffers

Consider using the following protocol:
Dilute 100 μg protein in 250 μL solution of 8 M urea + 0.5 M ß-mercaptoethanol, pH 7.5 adjusted with 1 M Tris.
Incubate at +37 °C overnight.
Dialyze against 20 mM iodine acetamide + 50 mM K-phosphate.
Dialyze against 50 mM K-phosphate, pH 7.5 (4 x 2 h).
Add 12 U enzyme, digest overnight at +37 °C.

Troubleshooting

Modified Incubation Conditions

The enzyme is unstable in higher concentrations of both urea and guanidinium-HCl, but it is possible to use as substrate a glycoprotein which was denatured with urea.
A procedure to remove the urea is given in ′Protocols′.
DTT does not interfere, independently of the concentration.

Materials
Loading
Sign In To Continue

To continue reading please sign in or create an account.

Don't Have An Account?