Recombinant A. tubingensis PNGase Protein, CF

R&D Systems | Catalog # 9586-GH

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Key Product Details

  • R&D Systems Sf 21 (baculovirus)-derived Recombinant A. tubingensis PNGase Protein (9586-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

Sf 21 (baculovirus)

Accession Number

OJI88926.1

Applications

Enzyme Activity
View all PNGase At Proteins and Enzymes »

Product Specifications

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

Purity

>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

His

Predicted Molecular Mass

60 kDa

SDS-PAGE

72-85 kDa, reducing conditions

Activity

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.

Formulation, Preparation, and Storage

9586-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 At

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).

References

  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.

Long Name

Peptide-N4-(N-acetyl-beta-D-glucosaminyl asparagine amidase)

UniProt

OJI88926.1

Additional PNGase At Products

Product Documents for Recombinant A. tubingensis PNGase 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 A. tubingensis PNGase Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant A. tubingensis PNGase Protein, CF (9586-GH):

Materials
  •  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

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