Recombinant F. meningosepticum PNGase F Protein, CF
Recombinant F. meningosepticum PNGase F Protein, CF Summary
Ala41-Asn354 with N-terminal Met and 6-His tag
|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
Recombinant F. meningosepticum PNGase 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. The E. coli-produced enzyme from R&D Systems offers a better value than the competition
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.
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