Recombinant F. meningosepticum PNGase F Protein, CF
Recombinant F. meningosepticum PNGase F Protein, CF Summary
Why choose R&D Systems PNGase F Enzyme?
- Guaranteed Enzymatic Activity and High Purity: Enzymatic activity tested in a deglycosylation assay and purity determined by SDS-PAGE to be greater than 95%.
- Removes N-linked Sugars from Glycoproteins: See the PNGase F protocol and activity data below. Compare and save!
- Lot-to-Lot Consistency: Stringent QC testing performed on each lot to ensure consistent activity and purity.
- Bulk Quantities Available: Bulk up and save with large mass quantities to meet your research needs. Supply agreements available, partner with us. Please contact us.
- Most Respected, Most Cited Brand in Proteins: With over 35 years of providing the best recombinant proteins to the scientific community, R&D Systems continues to lead the industry in quality, activity, and purity.
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
Recombinant F. meningosepticum PNGase F Protein, CF (Catalog # 9109-GH) is an amidase that cleaves the amide bond between the GlcNAc residue of an N-glycan and the underlying asparagine residue. Only the pentasaccharide core of an N-glycan is pictured here for clarity. The pentasaccharide can be extended at R1, R2 and R3 positions. R1 can be an alpha -6 fucose but not an alpha -3 fucose. R2 and R3 can be any of common monosaccharides or oligosaccharides.
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.
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.
2 μg/lane of Recombinant F. meningosepticum PNGase F Protein (Catalog # 9109-GH) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 34 kDa.
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.
Citations 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.
Citations: Showing 1 - 3
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New monoclonal antibodies that recognize an unglycosylated, conserved, extracellular region of CD44 in vitro and in vivo, and can block tumorigenesis
Authors: DF Lusche, DJ Wessels, RJ Reis, CC Forrest, AR Thumann, DR Soll
PLoS ONE, 2021-04-23;16(4):e0250175.
Sample Types: Whole Cells
Alternative splicing and cleavage of GLUT8
Authors: CM Alexander, JA Martin, E Oxman, I Kasza, KA Senn, H Dvinge
Mol Cell Biol, 2020-12-21;0(0):.
Sample Types: Cell Lysates
Continuous Translation of Circularized mRNA Improves Recombinant Protein Titer
Authors: A Costello, NT Lao, N Barron, M Clynes
Metab. Eng., 2019-01-04;0(0):.
Species: Transgenic Hamster
Sample Types: Cell Lysates
Applications: Enzyme Assay
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