PNGase F(Glycerol-free)

Storage Solution : 40U/ul PNGase F、20 mM Tris-HCl、50 mM NaCl, 5 mM EDTA (pH 7.5 @ 25°C)

10*NP-40: 10% NP-40 in MilliQ-H2O

10*Denaturing Buffer: 5% SDS、400 mM DTT

10*Reaction Buffer: 500mM Tris-HCl (pH 7.5 @ 25°C)

1. Denaturing Reaction Conditions:

1. Combine 1-20 µg of glycoprotein, 1 µl of Denaturing Buffer (10X) and H2O (if necessary) to make a 10 µl total reaction volume.

2. Denaturation is terminated by heating to 100℃ for 10-20min and cooling to room temperature.

3. Make a total reaction volume of 20 µl by adding 2 µl Reaction Buffer (10×), 2 µl 10% NP-40 and 6 µl H2O.

4. Add 1 µl PNGase F, mix gently.

5. Enzymatic digestion at 37℃ for 1h

6. Analyze by the method of SDS-PAGE

2. Non-Denaturing Reaction Conditions:

1. Combine 1-20 µg of glycoprotein, 2 µl of Reaction Buffer (10×) and H2O (if necessary) to make a 20 µl total reaction volume.

2. Add 2-5 µl PNGase F, mix gently.

3. Enzymatic digestion at 37°C for 4 - 24 hours.

4. Analyze by the method of SDS-PAGE