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  • PNGase F

    Description

    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)

    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. This modification is most commonly found in plant and some insect glycoproteins.

    Product Source

    PNGase F is purified from Flavobacterium meningosepticum (3) and it is free of proteases and Endo F activities.

    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


    Glycoprotein analysis

    • Removal of high mannose, hybrid, and complex N-glycans from glycoproteins
    • Free of contaminants (Endo F, proteases, etc.)

    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

    -20°C

    Storage Conditions

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

    Heat Inactivation

    75°C for 10 min

    Molecular Weight

    Apparent: 36000 daltons

    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. Typical reaction conditions: Please see Protocols

    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 happens to the asparagine after PNGase removes the sugar?
    3. What are Glycosidases and their uses?
    4. 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?
    5. What are the typical reaction conditions for PNGase F?
    6. Do detergents inhibit exoglycosidases/endoglycosidases?
    7. Does PNGase F work in Urea?
    8. What is a good endoglycosidase substrate?
    9. How do I inhibit PNGase F?
    10. How much PNGase F should I use to remove my carbohydrate under native conditions?
    11. I tried the PNGase F on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
    12. What is the difference between PNGase F, Endo H and O-Glycosidase?

    Tech Tips

    PNGase F
    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

    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

    Citations

    • Stech M, Quast RB, Sachse R, Schulze C, Wüstenhagen DA, Kubick S (2014)A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells PLoS One 9(5), e96635. PubMedID: 24804975, DOI: 10.1371/journal.pone.0096635
    • Kwon HM, Lee KH, Han BW, Han MR, Kim DH, Kim DE (2014)An RNA aptamer that specifically binds to the glycosylated hemagglutinin of avian influenza virus and suppresses viral infection in cells PLoS One 9(5), e97574. PubMedID: 24835440, DOI: 10.1371/journal.pone.0097574
    • Haller G, Li P, Esch C, Hsu S, Goate AM, Steinbach JH (2014)Functional characterization improves associations between rare non-synonymous variants in CHRNB4 and smoking behavior PLoS One 9(5), e96753. PubMedID: 24804708, DOI: 10.1371/journal.pone.0096753
    • Wright CR, Brown EL, Della-Gatta PA, Ward AC, Lynch GS, Russell AP (2014)G-CSF does not influence C2C12 myogenesis despite receptor expression in healthy and dystrophic skeletal muscle Front Physiol 5, 170. PubMedID: 24822049, DOI: 10.3389/fphys.2014.00170
    • Wicht O, Burkard C, de Haan CA, van Kuppeveld FJ, Rottier PJ, Bosch BJ (2014)Identification and Characterization of a Proteolytically Primed Form of the Murine Coronavirus Spike Proteins after Fusion with the Target Cell J Virol 88(9), 4943-52. PubMedID: 24554652, DOI: 10.1128/JVI.03451-13
    • Rosenbaek LL, Kortenoeven ML, Aroankins TS, Fenton RA (2014)Phosphorylation decreases ubiquitylation of the thiazide-sensitive cotransporter NCC and subsequent clathrin-mediated endocytosis J Biol Chem 289(19), 13347-61. PubMedID: 24668812, DOI: 10.1074/jbc.M113.543710
    • Botto L, Cunati D, Coco S, Sesana S, Bulbarelli A, Biasini E, Colombo L, Negro A, Chiesa R, Masserini M, Palestini P (2014)Role of lipid rafts and GM1 in the segregation and processing of prion protein PLoS One 9(5), e98344. PubMedID: 24859148, DOI: 10.1371/journal.pone.0098344
    • Itahana Y, Han R, Barbier S, Lei Z, Rozen S, Itahana K (2014)The uric acid transporter SLC2A9 is a direct target gene of the tumor suppressor p53 contributing to antioxidant defense Oncogene PubMedID: 24858040, DOI: 10.1038/onc.2014.119
    • Rosenbaum EE, Vasiljevic E, Brehm KS, Colley NJ (2014)Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster PLoS Genet 10(5), e1004349. PubMedID: 24785692, DOI: 10.1371/journal.pgen.1004349
    • Möykkynen T, Coleman SK, Semenov A, Keinänen K (2014)The N-terminal domain modulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization J Biol Chem 289(19), 13197-205. PubMedID: 24652293, DOI: 10.1074/jbc.M113.526301
    • Arakel EC, Brandenburg S, Uchida K, Zhang H, Lin YW, Kohl T, Schrul B, Sulkin MS, Efimov IR, Nichols CG, Lehnart SE, Schwappach B (2014)Tuning the electrical properties of the heart by differential trafficking of KATP ion channel complexes J Cell Sci 127(Pt 9), 2106-19. PubMedID: 24569881, DOI: 10.1242/jcs.141440

    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 Data Sheets

    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.