Recombinant F. meningosepticum PNGase F Protein, CF

Recombinant F. meningosepticum PNGase F Protein, CF (Catalog # 9109-GH) is an amidase that cleaves the amide bond between the GlcNAc residue of an N-glycan and the underlying asparagine residue. Only the pentasaccharide core of an N-glycan is pictured here for clarity. The pentasaccharide can be extended at R1, R2 and R3 positions. R1 can be an alpha -6 fucose but not an alpha -3 fucose. R2 and R3 can be any of common monosaccharides or oligosaccharides.

Total activity per vial of Recombinant F. meningosepticum PNGase F (Catalog # 9109-GH) compared to the leading competitor. R&D Systems® PNGase F gives you 2x more enzyme at a comparable price.

Recombinant F. meningosepticum PNGase F (Catalog # 9109-GH) from R&D Systems and a leading competitor are able to deglycosylate 10 μg of RNase B at 37 °C in one hour. The E. coli-produced enzyme from R&D Systems offers a better value than the competition

2 μg/lane of Recombinant F. meningosepticum PNGase F Protein (Catalog # 9109-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 34 kDa.

• Reactivity: Fm
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant F. meningosepticum PNGase F Protein, CF Summary

• Additional Information: Effective removal of N-linked oligosaccharides from glycoproteins. Glycerol-free, suitable for downstream MS and HPLC.
• Details of Functionality: Measured by its ability to deglycosylate ribonuclease B under denatured conditions. >50% ribonuclease B (10 μg) is deglycosylated by 2.5 ng rFmPNGase F within 30 minutes, as measured under the described conditions.
• Source: E. coli-derived f. meningosepticum PNGase F protein
  Ala41-Asn354 with N-terminal Met and 6-His tag
• Accession #: P21163
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 36 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 34 kDa, under reducing conditions.

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 0.1 M Tris, pH 7.5
  • Denaturing Buffer (10X): 5% SDS, 0.8 M beta -Mercaptoethanol
  • Recombinant F. meningosepticum PNGase F (rFmPNGase) (Catalog # 9109-GH)
  • Ribonuclease B, from bovine pancreas (RNase B) (Sigma, Catalog # R7884), 2.5 mg/mL stock in 25 mM Tris, pH 7.5
  • 10% Triton® X-100 (Amresco, Catalog # M236)
  • Reducing SDS-PAGE Sample Buffer
  • SDS-PAGE or Western Blot
  1. Dilute Denaturing Buffer to 5X in deionized water.
  2. Create a Substrate Mixture containing 0.8 mg/mL RNase B and 1X Denaturing Buffer in deionized water.
  3. Heat Substrate Mixture at 100 °C for 10 minutes. Cool to room temperature and microcentrifuge briefly.
  4. Add 10% Triton® X-100 to a final concentration of 1.67%.
  5. Dilute rFmPNGase F to 0.167 ng/µL in Assay Buffer.
  6. Combine 15 µL of Substrate Mixture and 15 µL 0.167 ng/µL rFmPNGase F. Include a control containing 15 µL of Substrate Mixture and 15 µL of Assay Buffer.
  7. Incubate reaction mixture at 37 °C for 30 minutes.
  8. Combine equal volumes of incubated reaction mixture and reducing SDS-PAGE sample buffer and boil samples at 100 °C for
     3-5 minutes.
  9. Load 15 µL (2.5 µg RNase B) per lane on a 4-20% SDS-PAGE gel.
  10. Stain gel and analyze for percent deglycosylation using densitometry.
  Per Reaction:
  • rFmPNGase F: 2.5 ng
  • RNase B: 10 µg

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant F. meningosepticum PNGase F Protein, CF

• PNGase F
• PNGF

Background

PNGase F, peptide N-glycosidase F from Flavobacterium meningosepticum, catalyzes the hydrolysis of asparagine-linked high mannose, as well as hybrid and complex oligosaccharides from glycoproteins (1). Unlike glycosidases that hydrolyze glycosidic bonds, PNGase F is an amidase that cleaves the
beta-aspartylglucosamine bond between the innermost GlcNAc of N-glycans and asparagine residues of glycoproteins (2). The enzyme is highly active on various
N-glycans except those with the innermost GlcNAc modified with alpha 1-3-linked core fucose, which is commonly found on plant glycoproteins (3). Cleavage with PNGase F will convert the asparagine residue to an aspartic residue, allowing identification of the glycosylation site by mass spectrometry (4). This purified enzyme is compatible with glycan analysis using mass spectrometry.

1. Elder, J.H. and Alexander, S. (1982) Proc. Natl. Acad. Sci. USA 79:4540.
2. Maley, F. et al. (1989) Anal. Biochem. 180:195.
3. Tarentino, A.L. and Plummer, T.H. (1994) Methods Enzymol 230:44.
4. Zhang, H. et al. (2003) Nat. Biotechnol. 21:660.

Publications for PNGase F (9109-GH)(3)

Publications using 9109-GH

• DF Lusche, DJ Wessels, RJ Reis, CC Forrest, AR Thumann, DR Soll New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis PLoS ONE, 2021-04-23;16(4):e0250175. 2021-04-23 [PMID: 33891595] (Bioassay, Human)
• CM Alexander, JA Martin, E Oxman, I Kasza, KA Senn, H Dvinge Alternative splicing and cleavage of GLUT8 Mol Cell Biol, 2020-12-21;0(0):. 2020-12-21 [PMID: 33077497] (Bioassay, Human)
• A Costello, NT Lao, N Barron, M Clynes Continuous Translation of Circularized mRNA Improves Recombinant Protein Titer Metab. Eng., 2019-01-04;0(0):. 2019-01-04 [PMID: 30615942] (Enzyme Assay, Transgenic Hamster)

Bioinformatics

• Uniprot:
  • P21163()