Recombinant F. meningosepticum PNGase F' (CystatinA Tag), CF
Recombinant F. meningosepticum PNGase F' (CystatinA Tag), CF Summary
|Met||6-His tag||Cystatin A
Accession # P01040
Accession # AAA85323
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, NaCl and EDTA.|
|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
- Substrate Denaturing Buffer (10X): 5% (w/v) SDS, 0.8 M beta -Mercaptoethanol
- Recombinant F. meningosepticum PNGase F' (Cystatin A Tag) (rFmPNGase F') (Catalog # 7695-GHP)
- 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, Peroxide Free (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 Substrate 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 2.0 µg/mL in Assay Buffer.
- Combine 15 µL of Substrate Mixture and 15 µL of 2.0 µg/mL rFmPNGase F'. Include a control containing 15 µL of Substrate Mixture and 15 µL of Assay Buffer.
- Incubate mixture at 37 °C for 30 minutes.
- Combine equal volumes of incubated reaction mixture and reducing SDS-PAGE sample buffer and boil samples at 100 °C for 3‑5 minutes.
- 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': 30 ng
- RNase B: 10 µg
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 sites by mass spectrometry (4). Recombinant PNGase F’ is a fusion of human cystatin A to PNGase F (Catalog # 7695-GH). PNGase F’ is a good alternative for PNGase F for deglycosylating proteins that have similar mass to PNGase F.
- 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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