Ludger PNGase F enzyme structure

    PNGase F (Peptide N Glycosidase F)

    PNGase F is suitable for release of all types (high-mannose, hybrid and complex) N-linked glycans from glycoproteins and glycopeptides. PNGase F will not remove oligosaccharides containing α(1-3) linked core fucose commonly found on plant glycoproteins.


    E-PNG01


      View product documentation: Specsheet / CofA / MSDS

    • Part Number
    • E-PNG01
    • Amount of Enzyme
    • 0.3 U / 60 µL








    Kit includes enzyme plus reaction buffers.
    Sufficient for up to 60 reactions.





    E-PNG01-200    *formerly E-PNG05


      View product documentation: Specsheet / CofA / MSDS

    • Part Number
    • E-PNG01-200
    • Amount of Enzyme
    • 1 U / 200 µL








    Includes enzyme only.
    Sufficient for up to 200 reactions.






    Product Specifications:


    PNGase F cleaves N-linked (asparagine-linked) oligosaccharides from glycoproteins. The enzyme deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. The enzyme will remain fully active under reaction conditions (37°C) for at least 96 hours. PNGase F will not remove oligosaccharides containing Alpha-(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A.

    There are a number of alternative enzymes which can be used to remove N-glycans, most especially the Endo F family of enzymes and Endo H. These enzymes cleave between the two N-acetylglucosamine residues in the core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. This leaves a charged sugar which can assist in keeping proteins in solution that precipitate after deglycosylation with PNGase F which removes the oligosaccharide intact. Endo F1 cleaves high mannose and some hybrid type N-glycans. Endo F2 will removes biantennary and high mannose (at a 40X reduced rate). Endo F3 releases of triantennarry and fucosylated biantennary N-glycans. Endo H removes hybrid or high mannose glycans.

    Source: Elizabethkingia miricola (was Chryseobacterium meningosepticum)

    EC: 3.5.1.52

    Alternative Names: PNGase F, Peptide N Glycosidase F, N-Glycosidase, N-Glycanase

    Contents E-PNG01:
    Ludger PNGase F - Kit contents
    PNGase F in 20 mM Tris-HCl, pH 7.5 - 60µL
    5x Reaction Buffer 7.5 – 250 mM sodium phosphate, pH 7.5
    Denaturation Solution – 2% SDS, 1 M Beta-mercaptoethanol
    Triton X-100 – 15% solution

    Contents E-PNG01-200 (enzyme only):
    PNGase F in 20 mM Tris-HCl, pH 7.5 - 200µL

    Specific Activity: >25 U/mg

    Activity: 5 U/mL

    Molecular weight: 36,000 daltons

    pH range: 6-10, optimum 7.5

    Protocol:
    1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 35 µL final volume with de-ionized water.
    2. Add 10 µL 5x Reaction Buffer 7.5 and 2.5 µL of Denaturation Solution. Heat at 100°C for 5 minutes.
    3. Cool. Add 2.5 µL of Triton X-100 and mix.
    4. Add 2.0 µL of enzyme to the reaction. Incubate 3 hours at 37°C.

    Specificity: Cleaves all asparagine-linked complex, hybrid or high mannose oligosaccharides unless alpha(1-3) core fucosylated; asparagine must be peptide bonded at both termini, Endo F free

    Specific Activity: Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured RNase B in 1 minute at 37°C, pH 7.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster).

    Storage: Store enzyme at 4°C.





Companion Products:

Process controls


Glycan clean-up - removal of proteins and enzymes from glycan mixtures following endoglycosidase treatment

  • LC-PBM-96
  • LudgerClean Protein Binding Membrane (96 samples)

Labelling and derivatisation of released glycans - View our Glycan Labelling Summary Table

  • LT-KAB-A2
  • LT-KAB-VP24
  • LT-KAB-VP96
  • LT-KPROC-24
  • LT-KPROC-96
  • LT-KPROC-VP24
  • LT-KAA-A2
  • LT-KAA-VP24
  • LT-PERMET-96
  • LT-PERMET-VP96
  • LudgerTag 2-AB Glycan Labelling Kit, sodium cyanoborohydride (24 samples)
  • LudgerTag 2-AB Glycan Labelling Kit, picoline borane (24 samples)
  • LudgerTag 2-AB Glycan Labelling Kit, picoline borane (96 samples)
  • LudgerTag Procainamide Glycan Labelling Kit, sodium cyanoborohydride (24 samples)
  • LudgerTag Procainamide Glycan Labelling Kit, sodium cyanoborohydride (96 samples)
  • LudgerTag Procainamide Glycan Labelling Kit, picoline borane (24 samples)
  • LudgerTag 2-AA Glycan Labelling Kit, sodium cyanoborohydride (24 samples)
  • LudgerTag 2-AA Glycan Labelling Kit, picoline borane (24 samples)
  • LudgerTag Permethylation Kit (96 samples)
  • LudgerTag Permethylation Kit, without methyl iodide (96 samples)

Alternative enzymes

  • E-EF01
  • E-EF02
  • E-EF03
  • E-EH02
  • E-G001
  • KE-DG01
  • KE-DGMX
  • Endoglycosidase F1
  • Endoglycosidase F2
  • Endoglycosidase F3
  • Endoglycosidase H
  • O-glycosidase
  • Enzymatic CarboRelease Kit - contains enzymes necessary for removing N-linked and O-linked oligosaccharides
  • Enzymatic DeGlycoMx Kit - contains enzyme mix for removing N-linked and O-linked oligosaccharides






References:

1. Bayer, E.A., F. De Meester, T. Kulik and M. Wilchek. Preparation of deglycosylated egg white avidin. Appl Biochem Biotech 53: 1-9 (1995)

2. Elder, J.H. and S. Alexander. endo-b-N-Acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proc Natl Acad Sci USA 79: 4540-4544 (1982)

3. Tarentino, A .L., C.M. Gomez and T.H. Plummer, Jr. Deglycosylation of asparagine-linked glycans by peptide :N-glycosidase F. Biochemistry 24: 4665-4671 (1985)

4. Tarentino A.L. and T.H. Plummer. Enzymatic deglycosylation of asparagine -linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Meth Enzymol 230: 44-57 (1994)

5. Trimble R.B. and A.L. Tarentino. Identification of distinct endoglycosidase (endo) activities in Flavobacterium meningosepticum: endo F1 , endo F2 and endo F3. Endo F1 and endo H hydrolyze only high mannose and hybrid glycans. J Biol Chem 266: 1646-1 651 (1991)

6. Taga, E. M., A. Waheed and R. L. Van Etten. Structural and chemical characterization of a homogeneous peptide-N-glycosidase from almond. Biochemistry 23: 815-22 (1984)

7. Tarentino AL, Trimble RB, Plummer TH. Enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins. Methods in Cell Biology: 32: 111-39 (1989)

8. Anthony L. , Tarentino and Thomas H. Plummer Jr. Enzymatic deglycosylation of asparagine-linked glycans: Purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Methods in Enzymology: 230: 44-57. (1994)

9. Tarentino AL, Plummer TH. Oligosaccharide accessibility to peptide:N-glycosidase as promoted by protein-unfolding reagents. The Journal of Biological Chemistry. 257 (18): 10776-80. (1982)