Recombinant F. meningosepticum PNGase F Protein, CF

Suitable for 8,000 (20 ug size) or 40,000 (100 ug size) RNase B (10 ug) deglycosylation reactions using our protocol.
  
  • 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.
  • 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.
  • Source
    E. coli-derived Ala41-Asn354 with N-terminal Met and 6-His tag
  • Accession #
  • N-terminal Sequence
    Analysis
    Met
  • Predicted Molecular Mass
    36 kDa
  • SDS-PAGE
    34 kDa, reducing conditions
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.
Assay Procedure
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
Data Images
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
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:
    PNGase F; PNGF
Related Research Areas
Primary Antibodies
Description Application Cat# Citations Images  

His Tag Antibody

WB, Simple Western, Flow, AP, CyTOF-ready MAB050 26  
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His Tag HRP-conjugated Antibody

WB, Simple Western MAB050H 3  
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His Tag APC-conjugated Antibody

ICFlow IC050A  
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His Tag Biotinylated Antibody

WB BAM050 4  
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His Tag PE-conjugated Antibody

ICFlow IC050P 2  
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His Tag Antibody

WB, Flow, AP MAB050R  
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His Tag Alexa Fluor® 488-conjugated Antibody

ICFlow IC050G  
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His Tag PerCP-conjugated Antibody

ICFlow IC050C  
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His Tag Alexa Fluor® 700-conjugated Antibody

ICFlow IC050N  
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Protein Purification Kits
Description Application Cat# Citations Images  

Histidine-tagged Protein Purification Resin

IP999 1
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