PNGase F (C09-1017-527)

Product DescriptionPNGase F cleaves N-linked (asparagine-linked) oligosaccharides from glycoproteins. The enzyme deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. PNGase F will not remove oligosaccharides containing Alpha-(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A.Denaturation increases the rate of cleavage. Most native proteins can still be completely N-deglycosylated but incubation time may need to be increased. The enzyme will remain fully active under reaction conditions (37˚C) for at least 96 hours.There are a number of alternative enzymes which can be used to remove N-glycans, most especially the Endo F family of enzymes and Endo H. These enzymes cleave between the two N-acetylglucosamine residues in the core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. This enhances the solubility, keeping proteins in solution that precipitate after deglycosylation with PNGase F which removes the oligosaccharide intact. Endo F1cleaves high mannose and some hybrid type N-glycans. Endo F2 will removes biantennary and high mannose (at a 40X reduced rate). Endo F3releases of triantennarry and fucosylated biantennary N-glycans. Endo H removes hybrid or high mannose glycans.Contents PNGase F in 20 mM Tris-HCl, pH 7.5Included with 20 µL and 60 µL pack sizes:5x Reaction Buffer 7.5 – 250 mM sodium phosphate, pH 7.5Denaturation Solution – 2% SDS, 1 M Beta-mercaptoethanolTriton X-100 – 15% solutionSpecific Activity >25 U/mgActivity 5 U/mlMolecular weight 35,000 daltonspH range 6-10, optimum 7.5PNGase F Protocol1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 35 µl final volume with de-ionized water.2. Add 10 µl 5x Reaction Buffer 7.5 and 2.5 µl of Denaturation Solution. Heat at 100°C for 5 minutes.3. Cool. Add 2.5 µl of Triton X-100 and mix.4. Add 2.0 µl of enzyme to the reaction. Incubate 3 hours at 37°C.Specifictity Cleaves all asparagine-linked complex, hybrid or high mannose oligosaccharides unless alpha(1-3) core fucosylated; asparagine must be peptide bonded at both termini, Endo F freeSpecific ActivityDefined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured RNase B in 1 minute at 37°C, pH 7.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster). Specifications and Purity: Specific Activity >25 U/mg;Activity 5 U/ml. Related Document: https://ald-pub-files.oss-cn-shanghai.aliyuncs.com/aladdinsci/pdp/sds/1/P420186-SCI_39a44bc9f91d2e6ff2b8e6583fae9154.pdf.