Recombinant A. tubingensis PNGase Protein, CF

Catalog #: 9586-GH Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
9586-GH-020
9586-GH-100

R&D Systems Recombinant Proteins and Enzymes

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant A. tubingensis PNGase Protein, CF Summary

Product Specifications

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.

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.

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
Analysis

His

Predicted Molecular Mass

60 kDa

SDS-PAGE

72-85 kDa, reducing conditions

Product Datasheets

9586-GH

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

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.

Assay Procedure

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

Create a Substrate Mixture containing 0.3 mg/mL RNase B and 1X Denaturing Buffer in Assay Buffer.
Heat Substrate Mixture at 100 °C for 5 minutes. Cool to room temperature and microcentrifuge briefly.
Dilute 10% Triton® X-100 to 1% in Assay Buffer.
Combine equal volumes of 1% Triton® X-100 and Substrate Mixture.
Dilute rA.t PNGase to 50 ng/µL in Assay Buffer.
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.
Incubate mixture at 37 °C for 120 minutes.
Add 20 µL of reducing SDS-PAGE Sample Buffer to incubated reaction mixture and boil samples at 100 °C for 3-5 minutes.
Load 35 µL (3 µg RNase B) per lane on a 4-20% SDS-PAGE gel.
Stain gel and analyze for percent deglycosylation using densitometry.

Per Reaction:

A.tubingensis PNGase: 500 ng
RNase B: 6 µg

Reconstitution Calculator

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