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    PNGase F (Glycerol-free)

    Description

    Peptide: N-Glycosidase F, also known as PNGase F, is an amidase and 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.).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Glycoprotein Denaturing Buffer-2010X
    NP-40-2010%
    GlycoBuffer 2-2010X

    Advantages and Features

    Applications

    • Removal of N-linked glycans from glycoproteins
    • Preferred formulation for HPLC and MS intensive methods of glycoprotein analysis

    Properties and 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 (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 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

    1X NP-40
    1% NP-40 in MilliQ-H2O

    Storage Temperature

    4°C

    Storage Conditions

    20 mM Tris-HCl
    50 mM NaCl
    5 mM Na2EDTA
    pH 7.5 @ 25°C

    Heat Inactivation

    75°C for 10 min

    Molecular Weight

    Apparent: 36000 daltons

    Storage Notes

    • Do not freeze

    Notes

    1. 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.
    2. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required. 
    3. PNGase F will not cleave N-linked glycans containing core α1-3 Fucose. 
    4. Repeated freeze thaw cycles degrade enzyme activity over time. 
    5. Typical reaction conditions: Please see Protocol tab

    References

    1. Maley, F. et al. (1989). Anal. Biochem.. 180, 195-204.
    2. Tretter, V. et al. (1991). Eur. J. Biochem.. 199, 647-652.
    3. Plummer, T.H. Jr. and Tarentino, A.L. (1991). Glycobiology. 1, 257-263.

    FAQs

    1. Is PNGase F compatible with downstream analysis such as HPLC and Mass Spectrometry?
    2. What are Glycosidases and their uses?
    3. Why is my immunoprecipitated (IP) protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein?
    4. Do detergents inhibit exoglycosidases/endoglycosidases?
    5. Does PNGase F work in Urea?
    6. What is a good endoglycosidase substrate?
    7. How do I inhibit PNGase F?
    8. How much PNGase F should I use to remove my carbohydrate under native conditions?
    9. What is the difference between PNGase F, Endo H and O-Glycosidase?
    10. I tried the PNGase F on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?

    Tech Tips

    Don’t freeze-thaw this enzyme.  
    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).

    Protocols

    1. PNGase F Protocol

    Datacards

    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 info@neb.com or fill out the Technical Support Form for appropriate document.

    Usage Guidelines & Tips

    Application Notes

    Quality Control

    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.

    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 info@neb.com.

    Datacards

    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 info@neb.com or fill out the Technical Support Form for appropriate document.