PNGase F II

Storage Solution: 5U/μL PNGase FII in 20 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 50% Glycerol (pH 7.5 @ 25°C)
10* PBS: 136 mM NaCl, 2.6mM KCl, 10 mM Na₂HPO₄, 2 mM‌ KH₂PO₄ (pH 7.4 @ 25°C)

Denaturing Reaction Conditions:
1. Combine 1-5 µg of glycoprotein, 1µl of 10*PBS and H2O (if necessary) in a total reaction volume of 10 µl.
2. Denature glycoprotein by heating reaction at 100°C for 10 minutes.
3. Chill denatured glycoprotein on ice and centrifuge 10 seconds.
4. Make a total reaction volume of 20 µl by adding1-2 µl PNGase F II,1 µl of PBS(10X)and 7-8µl H2O.
5. Incubate reaction at 37°C for 12-16 hour.

1.Reactions may be scaled-up linearly to accommodate larger reaction volumes.

2. Avoid repeated freezing and thawing.

3. We suggest using recombinant Avidin from maize, or horseradish peroxidase (HRP) as positive controls.

4. Glycoprotein denaturation must not contain SDS, which completely inhibits the enzymatic activity of PNGase F II.