Peptide -N-Glycosidase F, also known as PNGase F, is an amidase that cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins (1)
PNGase F is purified from Flavobacterium meningosepticum (3) and it is free of proteases and Endo F activities.
The following reagents are supplied with this product:
Store at (°C)
Glycoprotein Denaturing Buffer
G7 Reaction Buffer
Advantages and Features
Removal of high mannose, hybrid, and complex N-glycans from glycoproteins
Free of contaminants (Endo F, proteases, etc.)
Properties and Usage
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 (65 NEB units = 1 IUB milliunit).
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 G7 Reaction Buffer, 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
40 mM DTT
20 mM Tris-HCl 50 mM NaCl 5 mM Na2EDTA 50% Glycerol pH 7.5 @ 25°C
75°C for 10 min
Apparent: 36000 daltons
Quality Assurance Statement
No contaminating exoglycosidase or Endoglycosidase F1, F2 or F3 activity could be detected. No contaminating proteolytic activity could be detected.
Quality Control Assays
The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
Glycosidase Activity (TLC): The product is tested in multiple reactions, each containing a fluorescently-labeled oligosaccharide or glycopeptide. The reaction products are analyzed by TLC for digestion of substrate. No contaminating exoglycosidase or endoglycosidase activity is detected.
Protease Activity (SDS-PAGE):
The product is tested for protease activity by incubation with a standard mixture of proteins resulting in no detectable degradation of the proteins as determined by SDS-PAGE.
Protein Purity (SDS-PAGE): The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
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 Protocols
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.
Material Safety Datasheets
The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at firstname.lastname@example.org.
The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at email@example.com or fill out the Technical Support Form for appropriate document.
You can use this enzyme under native or denaturing conditions
Under native conditions we recommend adding more enzyme and using longer incubation times
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.
PNGase F will not cleave N-linked glycans containing core α1-3 Fucose (PNGase A must be used in this instance)
Enzyme activity varies at different temperatures: 37°C - 100%; 30°C - 100%; 23°C - 65%; 17°C - 40% and 3°C - 0%
A good positive control substrate is RNase B