PNGase F (CarboClip)
CarboClip is a recombinant peptide-N-Glycosidase F (PNGase F), from Elizabethkingia sp, overexpressed in E. coli that contains a hexahistidine tag in its C-terminus. It cleaves the glycosidic bond between the reducing end GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides on N-linked glycoproteins.
CarboClip is provided glycerol-free as a lyophilized powder containing Tris-HCl, NaCl, and Na2EDTA.
Applications
- N-glycan analysis of antibodies and glycoproteins
- Deglycosylation of membrane proteins on cells, viruses, and exosomes
- Removal of N-Linked Glycans for Native Protein Mass Spectrometry
Unit Definition: One unit is defined as the amount of enzyme required to remove > 95 % of the carbohydrate from 10 µg of denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 µL.
Unit Definition Assay: 10 µg of RNase B are denatured with glycoprotein denaturing buffer (0.5 % SDS, 40 mM DTT) at 100°C for 10 minutes. After the addition of NP-40 and buffer (50 mM Sodium Phosphate pH 7.5), two-fold dilutions of PNGase F (1 μL) are added and the reaction mixture is incubated for 1 hour at 37°C. The separation of reaction products is visualized by SDS-PAGE 12 % acrylamide and stained with Coomassie blue.
Storage and Stability:
Lyophilized powder stored at -80°C and -20°C is stable for more than 1 year, at room temperature stable for more than 10 days. Reconstituted protein should be stored at 4°C and is stable for 2-3 months.