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  • PNGase F, Recombinant

    Description

    Peptide: N-Glycosidase F, also known as PNGase F, is a recombinant amidase which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins (1).

    Product Source

    Cloned from Elizabethkingia miricola (formerly Flavobacterium meningosepticum) and expressed in E. coli (2).

    Specificity

    Reagents Supplied

    The following reagents are supplied with this product:

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

    Advantages and Features

    Applications

    • Removal of carbohydrate residues from proteins

    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).

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    75°C for 10 min

    Molecular Weight

    Apparent: 36000 daltons

    Unit Assay Conditions

    10 μg of RNase B are denatured with 1X Glycoprotein Denaturing Buffer at 100°C for 10 minutes. After the addition of NP-40 and GlycoBuffer 2, two-fold dilutions of PNGase F, Recombinant are added and the reaction mix is incubated for 1 hour at 37°C. Separation of reaction products are visualized by SDS-PAGE.

    Notes

    1. To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
    2. Since PNGase F, Recombinant activity is inhibited by SDS, it is essential to have NP-40 in the reaction mixture. It is not known why this non-ionic detergent counteracts the SDS inhibition at the present time.
    3. PNGase F, Recombinant will not cleave N-linked glycans containing core α1-3 Fucose.

    References

    1. Maley, F. et al. (1989). Anal. Biochem. 180, 195-204.
    2. Chen, M. New England Biolabs, Inc. Unpublished observation

    FAQs

    1. Is there a difference between PNGase F (P0704/P0705) and PNGase F, Recombinant (P0708/P0709)?
    2. I tried deglycosylating my glycoprotein with Remove-iT® PNGase F but did not see removal of the carbohydrate. What could be the problem?
    3. What happens to the asparagine after PNGase removes the sugar?
    4. Why is protein degraded? When I denature and add SDS all I see on my SDS-PAGE is a smear or no protein. Can a protease inhibitor cocktail be used in a PNGase F, Recombinant reaction?
    5. What is the difference between PNGase F, Endo H and O-Glycosidase?
    6. Does PNGase F work in Urea?
    7. What are the typical reaction conditions for PNGase F, Recombinant?
    8. How much PNGase F, Recombinant should I use to remove my carbohydrate under native or DTT denaturing conditions?
    9. How do I inhibit PNGase F, Recombinant?
    10. Is PNGase F, Recombinant compatible with downstream analysis such as HPLC and Mass Spectrometry?
    11. What are glycosidases and their uses?
    12. Do detergents inhibit exoglycosidases/endoglycosidases?
    13. Why have the NEB Glycosidase enzymes changed reaction buffers? What are the new reaction buffers and can I still use an enzyme with its old buffer? Where can I find the composition of the old buffers?
    14. What is a good endoglycosidase substrate?

    Tech Tips

    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)
    5. A good positive control substrate is RNase B

    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 endoglycosidase, exoglycosidase or 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.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    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.