PNGase F

* One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 10 μg of denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 μl.

一. 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

二. 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

1.The target protein should be in a solution compatible with Rapid PNGase F activity. Avoid buffers containing SDS, as it inhibits PNGase F. Common stabilizing reagents such as Tween, Triton X-100, NP-40, octyl glucoside and non-detergent sulfobetaine, as well as traces of organic solvents, can prevent optimal rapid deglycosylation.

2.N-linked glycans containing core α1-3 Fucose are not cleaved by PNGase F.

3.For deglycosylation of native glycoprotiens, increased incubation time and increased amount of enzyme may be needed.

4.Optimize reaction conditions for each substrate.

5.If a larger amount of glycoprotein is used, scale up reaction volumes accordingly.

6.Although this product has been optimized for the rapid removal of N-glycans from antibodies, it can be utilized with various other glycoproteins.