Recombinant A. tubingensis PNGase Protein, CF

• Reactivity: Hu, Fg
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant A. tubingensis PNGase Protein, CF Summary

• Details of Functionality: Measured by its ability to deglycosylate ribonuclease B under denatured conditions. 50% ribonuclease B (6 μg) is deglycosylated by 500 ng of Recombinant A.tubingensis PNGase within 120 minutes, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived a. tubingensis PNGase At protein
  Leu22-Ser557 (F78I, E352G, P381S, S406Q, T412S, S414F, S451T and S555T), with an N-terminal 6-His tag
• Accession #: OJI88926.1
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 60 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: 72-85 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 and NaCl.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining
• Assay Procedure:
  • Assay Buffer: 0.1 M Sodium Citrate, pH 3.5
  • 10X Denaturing Buffer (5% SDS, 0.8 M beta -Mercaptoethanol)
  • Recombinant A. tubingensis PNGase At (rA.t PNGase) (Catalog # 9586-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. Create a Substrate Mixture containing 0.3 mg/mL RNase B and 1X Denaturing Buffer in Assay Buffer.
  2. Heat Substrate Mixture at 100 °C for 5 minutes. Cool to room temperature and microcentrifuge briefly.
  3. Dilute 10% Triton® X-100 to 1% in Assay Buffer.
  4. Combine equal volumes of 1% Triton® X-100 and Substrate Mixture.
  5. Dilute rA.t PNGase to 50 ng/µL in Assay Buffer.
  6. Combine 40 µL of Substrate Mixture and 10 µL of 50 ng/µL rA.t PNGase. Include a control containing 40 µL of Substrate Mixture and 10 µL of Assay Buffer.
  7. Incubate mixture at 37 °C for 120 minutes.
  8. Add 20 µL of reducing SDS-PAGE Sample Buffer to incubated reaction mixture and boil samples at 100 °C for 3-5 minutes.
  9. Load 35 µL (3 µg RNase B) per lane on a 4-20% SDS-PAGE gel.
  10. Stain gel and analyze for percent deglycosylation using densitometry.
  Per Reaction:
  • A.tubingensis PNGase: 500 ng
  • RNase B: 6 µg

Notes

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

Alternate Names for Recombinant A. tubingensis PNGase Protein, CF

• PNGase At

Background

Glycoamidases are used extensively to deglycosylate asparagine-linked glycoproteins to obtain intact N-glycans and core proteins, thus are vital for scientists to understand the structure and function of these glycoproteins. A well known glycoamidase is peptide-N4-(N-acetyl-beta -D-glucosaminyl) asparaginase amidase F (PNGase F), an enzyme native to Flavobacterium meningosepticum (1). Another glycoamidase, native to fungus Aspergillus tubingensis, called PNGase At, has similar function but distinct substrate specificity coverage. Compared to PNGase F, PNGase At has broader substrate specificity, wider pH activity curve, and can deglycosylate those N-glycans with alpha 1-3 fucosylated core structure, which is totally resistant to PNGase F (2).

1. Elder, J.H. and Alexander, S. (1982) Proc. Natl. Acad. Sci. USA 79:4540.
2. Ftouchi-Paquin, N. et al. (1997) J. Biol. Chem.  36:22960.

Bioinformatics

• Uniprot:
  • OJI88926.1()