Recombinant F. meningosepticum PNGase F Protein, CF
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Terms and Conditions
Recombinant F. meningosepticum PNGase F Protein, CF Summary
Ideal for removing all N-linked carbohydrates from glycoproteins
Ala41-Asn354 with N-terminal Met and 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 Tris, pH 7.5
- Denaturing Buffer (10X): 5% SDS, 0.8 M beta -Mercaptoethanol
- Recombinant F. meningosepticum PNGase F (rFmPNGase) (Catalog # 9109-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
- Dilute Denaturing Buffer to 5X in deionized water.
- Create a Substrate Mixture containing 0.8 mg/mL RNase B and 1X Denaturing Buffer in deionized water.
- Heat Substrate Mixture at 100 °C for 10 minutes. Cool to room temperature and microcentrifuge briefly.
- Add 10% Triton® X-100 to a final concentration of 1.67%.
- Dilute rFmPNGase F to 0.167 ng/µL in Assay Buffer.
- Combine 15 µL of Substrate Mixture and 15 µL 0.167 ng/µL rFmPNGase F. Include a control containing 15 µL of Substrate Mixture and 15 µL of Assay Buffer.
- Incubate reaction mixture at 37 °C for 30 minutes.
equal volumes of incubated reaction mixture and reducing SDS-PAGE
sample buffer and boil samples at 100 °C for
- Load 15 µL (2.5 µg RNase B) per lane on a 4-20% SDS-PAGE gel.
- Stain gel and analyze for percent deglycosylation using densitometry.
- rFmPNGase F: 2.5 ng
- RNase B: 10 µg
RecombinantF. meningosepticumPNGase F (Catalog # 9109-GH) from R&D Systems and a leading competitor are able to deglycosylate 10 μg of RNase B at 37 °C in one hour. TheE. coli-produced enzyme from R&D Systems offers a better value than the competition
Total activity per vial of Recombinant F. meningosepticum PNGase F (Catalog # 9109-GH) compared to the leading competitor. R&D Systems®PNGase F gives you 2x more enzyme at a comparable price.
Background: PNGase F
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 site by mass spectrometry (4). This purified enzyme is compatible with glycan analysis using mass spectrometry.
- Elder, J.H. and Alexander, S. (1982) Proc. Natl. Acad. Sci. USA 79:4540.
- Maley, F. et al. (1989) Anal. Biochem. 180:195.
- Tarentino, A.L. and Plummer, T.H. (1994) Methods Enzymol 230:44.
- Zhang, H. et al. (2003) Nat. Biotechnol. 21:660.
Citation for Recombinant F. meningosepticum PNGase F Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
Continuous Translation of Circularized mRNA Improves Recombinant Protein Titer
Authors: A Costello, NT Lao, N Barron, M Clynes
Metab. Eng., 2019;0(0):.
Species: Transgenic Hamster
Sample Types: Cell Lysates
Applications: Enzyme Assay
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