Description
Recombinant PNGase F is isolated from a E. coli strain containing a clone of the Elizabethkingia miricola gene. There is no detectable difference in activity or specific activity of the recombinant enzyme from the native enzyme.PNGase F cleaves asparagine-linked (N-linked) oligosaccharides from glycoproteins. PNGase F deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage up to 100x. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. PNGase F will remain active under incubation conditions for at least 72 hours. 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.Contents60 µl aliquot of recombinant PNGase F (0.3 U) in 20 mM Tris-HCl, pH 7.5Included with 20 µL and 60 µL pack sizes5x PNGase F Reaction Buffer 7.5 for PNGase F – 250 mM sodium phosphate, pH 7.5PNGase F Denaturation Solution – 2% SDS, 1 M Beta-mercaptoethanolPNGase F Triton X-100 – 15% solutionFormulationThe enzyme is provided as a sterile-filtered solution in 20 mM Tris-HCl (pH 7.5).Molecular Weighapproximately 35 kD.pH optimum7.5, active over the range 6-10.Specific ActivitySpecific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of RNase B in 1 minute at 37˚C, pH 7.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster).SpecificityPNGase F cleaves asparagine-linked (N-linked) oligosaccharides from glycoproteins. PNGase F deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage up to 100x. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. PNGase F will remain active under incubation conditions for at least 72 hours. 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. StabilitySeveral days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly. Quality & PurityPNGase F is tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The production host strain has been extensively tested and does not produce any detectable glycosidases.PNGase F Suggested usage1. 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 PNGase F Reaction Buffer 7.5 and 2.5 µl of PNGase F Denaturation Solution. Heat at 100˚C for 5 minutes.3. Cool. Add 2.5 µl of PNGase F Triton X-100 and mix.4. Add 2.0 µl of PNGase F to the reaction. Incubate 3 hours at 37˚C.If SDS or heat denaturation is omitted, increase incubation time to at least 24 hours. Monitor cleavage by SDS-PAGE