Recombinant A. tubingensis PNGase Protein, CF Summary
Leu22-Ser557 (F78I, E352G, P381S, S406Q, T412S, S414F, S451T and S555T), with an N-terminal 6-His tag
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.
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.
|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.
- 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.
- A.tubingensis PNGase: 500 ng
- RNase B: 6 µg
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).
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