Recombinant F. meningosepticum PNGase F' (CystatinA Tag), CF

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

Recombinant F. meningosepticum PNGase F' (CystatinA Tag), CF Summary

• Details of Functionality: Measured by its ability to deglycosylate ribonuclease B under denatured conditions. >50% ribonuclease B (10 μg) is deglycosylated by 30 ng of Recombinant F. meningosepticum PNGase F' within 30 minutes, as measured under the described conditions.
• Source: E. coli-derived f. meningosepticum PNGase F protein
  

  Met	6-His tag	Cystatin A (Met1-Phe98) Accession # P01040	Linker	PNGase F (Ala41-Asn354) Accession # AAA85323
  N-terminus				C-terminus

• Accession #: AAA85323 (PNGase F') & P01040 (Cystatin A)
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 48 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: 44-48 kDa, 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, NaCl and EDTA.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 0.1 M Tris, pH 7.5
  • Substrate Denaturing Buffer (10X): 5% (w/v) SDS, 0.8 M beta -Mercaptoethanol
  • Recombinant F. meningosepticum PNGase F' (Cystatin A Tag) (rFmPNGase F') (Catalog # 7695-GHP)
  • 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, Peroxide Free (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 Substrate 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 2.0 µg/mL in Assay Buffer.
  6. Combine 15 µL of Substrate Mixture and 15 µL of 2.0 µg/mL rFmPNGase F'. Include a control containing 15 µL of Substrate Mixture and 15 µL of Assay Buffer.
  7. Incubate 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': 30 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' (CystatinA Tag), 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 sites by mass spectrometry (4). Recombinant PNGase F’ is a fusion of human cystatin A to PNGase F (Catalog # 7695-GH) . PNGase F’ is a good alternative for PNGase F for deglycosylating proteins that have similar mass to PNGase F.

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.

Bioinformatics

• Uniprot:
  • AAA85323 (PNGase F') & P01040 (Cystatin A)()