Feb 22, 2022
Version 2
PNGase F Protocol (Non-Denaturing Reaction Conditions) V.2
- 1New England Biolabs
External link: https://www.neb.com/protocols/2014/07/31/pngase-f-protocol
Protocol Citation: New England Biolabs 2022. PNGase F Protocol (Non-Denaturing Reaction Conditions). protocols.io https://dx.doi.org/10.17504/protocols.io.be74jhqwVersion created by New England Biolabs
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: April 16, 2020
Last Modified: February 22, 2022
Protocol Integer ID: 35804
Keywords: pngasef, Essentials of Glycobiology, PNGase F is inhibited, deglycosylation, PNGase F denaturing reaction conditions, PNGase F non-denaturing reaction conditions
Abstract
PNGase F is the most effective enzymatic method for removing almost all N-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).
| A | B | C | D | E | F | G | H | |
| 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:
Non-Denaturing Reaction Conditions:
Non-Denaturing Reaction Conditions:
Note
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.
Combine 1 µg - 20 µg glycoprotein , 2 µL GlycoBuffer 2 (10X) and H2O (if necessary) to make a 20 µL total reaction volume.
Add 2 µL - 5 µL PNGase F , mix gently.
Incubate reaction at 37 °C for 04:00:00 - 24:00:00 .
Note
Note: To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
Analyze by method of choice.
Note
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.