PNGase F Protocol , Denaturing Conditions
Combine the following ( for a 10 µl total reaction volume ) :
Denature glycoprotein by heating reaction at 95°C for 5 minutes .
Chill denatured glycoprotein on ice .
Centrifuge 10 seconds .
Add the following ( bringing total reaction volume to 20 µl ) :
Add PNGase F , 1 µl .
Mix gently .
Incubate reaction at 37°C for 1 hour .
Analyze by method of choice
