PNGase F Procotol
PNGase F Procotol
Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and larger reaction volumes. Optimal incubation times may vary for particular substrates. Typical reaction conditions are as follows:
Denaturing Reaction Conditions:
1. Combine 1-20 µg of glycoprotein, 1 µl of 10X Glycoprotein Denaturing Buffer and H2O (if necessary) to make a 10 µl total reaction volume.
2. Denature glycoprotein by heating reaction at 95°C for 5 minutes.
3. Chill denatured glycoprotein on ice and centrifuge 10 seconds.
4. Make a total reaction volume of 20 µl by adding
1. 2 µl 10X GlycoBuffer 2,
2. 2 µl 10% NP40 and
3. 6 µl H2O.
PNGase F 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 F, 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.
2. Non-Denaturing Reaction Conditions:
When deglycosylating a native 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 non-denatured reaction can then be compared to the denatured reaction to determine the extent of reaction completion.
1. Combine 1-20 µg of glycoprotein, 2 µl of 10X GlycoBuffer 2 and H2O (if necessary) to make a 20 µl total reaction volume.
2. Add 2-5 µl PNGase F, mix gently.
3. Incubate reaction at 37°C for 4 - 24 hours.
Note: To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
4. Analyze by method of choice.
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.
Notes
If using P0704/P0708, we recommend limiting PNGase F to 1/10 (or less) of
Reactions may be scaled-up linearly to accommodate larger amounts of glycoprotein and larger reaction volumes. Optimal incubation times may vary for particular substrates. Typical reaction conditions are as follows:
Denaturing Reaction Conditions:
1. Combine 1-20 µg of glycoprotein, 1 µl of 10X Glycoprotein Denaturing Buffer and H2O (if necessary) to make a 10 µl total reaction volume.
2. Denature glycoprotein by heating reaction at 95°C for 5 minutes.
3. Chill denatured glycoprotein on ice and centrifuge 10 seconds.
4. Make a total reaction volume of 20 µl by adding
1. 2 µl 10X GlycoBuffer 2,
2. 2 µl 10% NP40 and
3. 6 µl H2O.
PNGase F 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 F, 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.
2. Non-Denaturing Reaction Conditions:
When deglycosylating a native 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 non-denatured reaction can then be compared to the denatured reaction to determine the extent of reaction completion.
1. Combine 1-20 µg of glycoprotein, 2 µl of 10X GlycoBuffer 2 and H2O (if necessary) to make a 20 µl total reaction volume.
2. Add 2-5 µl PNGase F, mix gently.
3. Incubate reaction at 37°C for 4 - 24 hours.
Note: To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
4. Analyze by method of choice.
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.
Notes
If using P0704/P0708, we recommend limiting PNGase F to 1/10 (or less) of