Feb 11, 2022
Version 2
PNGase F Protocol (Denaturing Conditions) V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: [email protected]
External link: https://www.neb.com/protocols/2014/07/31/pngase-f-protocol
Protocol Citation: New England Biolabs 2022. PNGase F Protocol (Denaturing Conditions). protocols.io https://dx.doi.org/10.17504/protocols.io.bikhkct6Version created by Liz Brydon
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: July 14, 2020
Last Modified: February 11, 2022
Protocol Integer ID: 39273
Keywords: Essentials of Glycobiology, glygoprotein, PNGase F is inhibited, deglycosylation, PNGase F denaturing reaction conditions, PNGase F non-denaturing reaction conditions, pngase f, linked oligosaccharide, effective enzymatic method, oligosaccharides from glycoprotein, complex oligosaccharide, glycoprotein, asparagine residues of high mannose, amidase, denaturing reaction condition
Abstract
PNGase F is the most effective enzymatic method for removing almost allN-linked oligosaccharides from glycoproteins. PNGase F is an amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides.
Guidelines
- For unit conversion between different suppliers, please reference the Glycobiology Unit Conversion Chart page.
Biology Unit Conversion Chart
Reagent companies differ in how a unit of enzyme is defined. This chart can be used to help determine how a unit of enzyme from one company compares to a unit of enzyme from NEB. All enzymes were assayed using NEB's assay protocols as a means of normalization (NEB Assay).
| Enzyme | Company | Selling Conc. (U/ml) | Units/Vial | µl/Vial | NEB Assay (U/ml) | NEB Assay Units /Vial | µl Conversion (1 NEB µl = x Company µls) | |
| PNGase F | NEB (NEB #P0704/P0705) | 500,000 | 15,000 | 30 | 500,000 | 15,000 | 1 | |
| Prozyme (GKE-5006A) | 2.5 | 0.1 | 40 | 150,000 | 6,000 | 3.3 | ||
| Prozyme (GKE-5020B, Ultra) | 10 | 0.4 | 40 | 500,000 | 20,000 | 1 | ||
| QA Bio (E-PNG01) | 5 | 0.3 | 60 | 200,000 | 12,000 | 2.5 | ||
| Sigma (P7367) | 500 | 50 | 50 | 90,000 | 4,500 | 5.5 |
Materials
MATERIALS
PNGase F (native) - 75,000 unitsNew England BiolabsCatalog #P0704L
PNGase F (native) - 15,000 unitsNew England BiolabsCatalog #P0704S
PNGase F Recombinant - 75,000 unitsNew England BiolabsCatalog #P0708L
PNGase F Recombinant - 15,000 unitsNew England BiolabsCatalog #P0708S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Before start
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:
Combine 1 µg - 20 µg glycoprotein , 1 µL Glycoprotein Denaturing Buffer (10X) and H2O (if necessary) to make a 10 µL total reaction volume.
Denature glycoprotein by heating reaction at 100 °C for 00:10:00 .
Chill denatured glycoprotein On ice and centrifuge 00:00:10 .
Make a total reaction volume of 20 µL by adding: 2 µL GlycoBuffer 2 (10X) , 2 µL 10% NP-40 and 6 µL H2O .
Note
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.
Add 1 µL PNGase F and mix gently.
Incubate reaction at 37 °C for 01:00:00 .
Analyze by method of choice.
Note
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.