Reaction Conditions for PNGase A (P0707)
Protocol
Denaturing Reaction Conditions:1. Combine 1-5 µg of glycoprotein, 1 µl of Glycoprotein Denaturing Buffer (10X) 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 adding 2 µl GlycoBuffer 3 (10X), 2 µl 10% NP-40 and 6 µl H2O.
Note: PNGase A is inhibited by SDS, therefore it is essential to have NP-40 in the reaction mixture under denaturing conditions. Failure to include NP-40 into the denaturing protocol will result in loss of enzymatic activity.
5. Add 1 µl PNGase A, mix gently.
6. Incubate reaction at 37°C for 1 hour.
7. Analyze by method of choice
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.
Reducing Reaction Conditions:
When deglycosylating a DTT reduced glycoprotein it is recommended that an aliquot of the glycoprotein is subjected to the denaturing protocol to provide a positive control for the fully deglycosylated protein. The DTT reduced reaction can then be compared to the denatured reaction to determine the extent of reaction completion.
1. Combine 1-5 µg of glycoprotein, 2 µl of GlycoBuffer 3 (10X), 4 µl of 0.2 M DTT and H2O (if necessary) to make a 20 µl total reaction volume.
2. Reduce glycoprotein by heating reaction at 55°C for 10 minutes.
3. Add 2-5 µl PNGase A, mix gently.
4. Incubate reaction at 37°C for 4 - 24 hours.
Note: To deglycosylate a DTT reduced glycoprotein, longer incubation time as well as more enzyme may be required.
5. Analyze by method of choice.
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.