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 ÃŸ-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.
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.