Recombinant F. meningosepticum PNGase F Protein, CF

R&D Systems | Catalog # 9109-GH

Effective removal of N-linked oligosaccharides from glycoproteins. Glycerol-free, suitable for downstream MS and HPLC.
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Key Product Details

  • R&D Systems E. coli-derived Recombinant F. meningosepticum PNGase F Protein (9109-GH)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P21163

Applications

Enzyme Activity
View all PNGase F Proteins and Enzymes »

Why choose R&D Systems PNGase F Enzyme?

  • Guaranteed Enzymatic Activity and High Purity: Enzymatic activity tested in a deglycosylation assay and purity determined by SDS-PAGE to be greater than 95%.
  • Removes N-linked Sugars from Glycoproteins: See the PNGase F protocol and activity data below. Compare and save!
  • Lot-to-Lot Consistency: Stringent QC testing performed on each lot to ensure consistent activity and purity.
  • Bulk Quantities Available: Bulk up and save with large mass quantities to meet your research needs. Supply agreements available, partner with us. Please contact us.
  • Most Respected, Most Cited Brand in Proteins: With over 35 years of providing the best recombinant proteins to the scientific community, R&D Systems continues to lead the industry in quality, activity, and purity.

Product Specifications

Source

E. coli-derived f. meningosepticum PNGase F protein
Ala41-Asn354 with N-terminal Met and 6-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

36 kDa

SDS-PAGE

34 kDa, under reducing conditions.

Activity

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.

Scientific Data Images for Recombinant F. meningosepticum PNGase F Protein, CF

Recombinant F. meningosepticum PNGase F Protein Enzyme Activity Diagram.

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.
Recombinant F. meningosepticum PNGase F Protein Bioactivity

Recombinant F. meningosepticum PNGase F Protein Bioactivity

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 Protein Enzyme Activity

Recombinant F. meningosepticum PNGase F Protein Enzyme Activity.

RecombinantF. meningosepticumPNGase 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. TheE. coli-produced enzyme from R&D Systems offers a better value than the competition.

Recombinant F. meningosepticum PNGase F Protein SDS-PAGE.

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.

Formulation, Preparation, and Storage

9109-GH
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Background: PNGase F

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.

References

  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.

Long Name

Peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine Amidase F

Alternate Names

PNGF

UniProt

P21163

Additional PNGase F Products

Product Documents for Recombinant F. meningosepticum PNGase F Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant F. meningosepticum PNGase F Protein, CF

For research use only

Citations for Recombinant F. meningosepticum PNGase F Protein, CF

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Protocols

View specific protocols for Recombinant F. meningosepticum PNGase F Protein, CF (9109-GH):

Materials
  • 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

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