PNGase F (N-Glycosidase F) is a recombinant glycosidase expressed and purified from E.coli which carries the Flavobacterium meningosepticum gene.
PNGase F cleaves between the innermost N-acetylglucosamine and asparagine residues of the chitobiose core of N-linked glycans, leaving only asparagine.PNGase F has a molecular weight of ~36.8 kDa.The optimal pH is 7.5.Substrate Specificity- Asn should be linked to at least one amino acid at the N- or C-terminal- hybrid N-linked oligosaccharides (except 1-3 linked fucose)Product Components- PNGase F : 100 ㎕ (500 unit/㎕)- 10x Reaction Buffer : 500 mM sodium citrate, pH 7.5.Unit Definition- One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 10 ㎍ of denatured RNase B for 1 hr at 37°C in a total reaction volume of 10 ㎕.Specific Activity- 500 unit/㎕, 1,000 unit/㎍Storage Buffer- 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, 50% glycerol.PNGase F Reaction Condition- 10 ㎍ of RNase B are denatured with 0.5% SDS, 1% β-mercaptoethanol at 100°C for 10 min. After the addition of Reaction Buffer to final 1x, PNGase F is added and the reaction mix is incubated for 1 hr at 37°C. Separation of reaction products is analyzed by SDS-PAGE (cleaved RNase B migrates faster). The activity of PNGase F is not affected by addition of 0.5% SDS. For a native glycoprotein, 5-fold more enzyme and longer incubation time will be required.Storage Temperature- Store at -20℃.QC Tests- Activity, SDS-PAGE purity, performance tests.
