PNGase F is the most effective enzymatic method for removing almost all N-linked oligosaccharides from
glycoproteins. PNGase F (Glycerol-free) is an amidase, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides.
Glycerol-free for optimal performance in HPLC and mass spectrometry analysis
≥ 95% purity, as determined by SDS-PAGE and intact ESI-MS
Non-recombinant with no detectable endoglycosidase F1, F2 or F3 contamination
Optimal activity and stability for up to 24 months
Can be used under native or denaturing conditions
Optimized for deglycosylation of glycoproteins; leaves N-glycan core oligosaccharides intact and suitable for further analysis
Peptide: N-Glycosidase F, also known as PNGase F, is an amidase which is supplied glycerol free for optimal performance in HPLC intensive methods. PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins (1).
Detailed Specificity PNGase F is not able to cleave N-linked glycans from glycoproteins when the innermost GlcNAc residue is linked to an α1-3 Fucose residue (2). This modification is most commonly found in plant and some insect glycoproteins.
Product Source
PNGase F is purified from Flavobacterium meningosepticum (3), free of contaminants (Endo F, proteases, etc.).
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
P0705S
4
PNGase F (Glycerol-free)
P0705SVIAL
4
1 x 0.03 ml
500,000 units/ml
GlycoBuffer 2
B3704SVIAL
-20
1 x 1 ml
10 X
Glycoprotein Denaturing Buffer
B1704SVIAL
-20
1 x 1 ml
10 X
NP-40
B2704SVIAL
-20
1 x 1 ml
10 %
P0705L
4
PNGase F (Glycerol-free)
P0705LVIAL
4
1 x 0.15 ml
500,000 units/ml
GlycoBuffer 2
B3704SVIAL
-20
1 x 1 ml
10 X
Glycoprotein Denaturing Buffer
B1704SVIAL
-20
1 x 1 ml
10 X
NP-40
B2704SVIAL
-20
1 x 1 ml
10 %
Properties & Usage
Unit Definition
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.
Unit Definition Assay: 10 µg of RNase B are denatured with 1X Glycoprotein Denaturing Buffer (0.5% SDS, 40 mM DTT) at 100°C for 10 minutes. After the addition of NP-40 and GlycoBuffer 2, two-fold dilutions of PNGase F are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE.
1X Glycoprotein Denaturing Buffer 0.5% SDS 40 mM DTT
Since PNGase F activity is inhibited by SDS, it is essential to have NP-40 present in the reaction mixture. Why this non-ionic detergent counteracts the SDS inhibition is unknown at present.
To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
PNGase F will not cleave N-linked glycans containing core α1-3 Fucose.
Typical reaction conditions: Please see Protocol tab
References
Maley, F. et al. (1989). Anal. Biochem.. 180, 195-204.
Tretter, V. et al. (1991). Eur. J. Biochem.. 199, 647-652.
Plummer, T.H. Jr. and Tarentino, A.L. (1991). Glycobiology. 1, 257-263.
1. You can use this enzyme under native or denaturing conditions.
2. Under native conditions we recommend adding more enzyme and using longer incubation times.
3. PNGase F activity is inhibited by SDS, therefore under denaturing conditions it is essential to have NP-40 present in the reaction mixture in a 1:1 ratio.
4. PNGase F will not cleave N-linked glycans containing core α1-3 Fucose (PNGase A must be used in this instance).
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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