Part number E-PNG01
Peptide N Glycosidase F, N-Glycosidase, PNGase F
QA-Bio™ 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.
PNGase F Source Elizabethkingia miricola (was Chryseobacterium meningosepticum)
60 µl aliquot of PNGase F (0.3 U) in 20 mM Tris-HCl, pH 7.5
5x PNGase F Reaction Buffer 7.5 for PNGase F - 250 mM sodium phosphate, pH 7.5
PNGase F Denaturation Solution - 2% SDS, 1 M Beta-mercaptoethanol
PNGase F Triton X-100 - 15% solution
Specific Activity >25 U/mg
Activity 5 U/ml
Molecular weight 36,000 daltons
pH range for PNGase F 6-10, optimum 7.5
PNGase F Suggested usage
1. 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.
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 free
Specific Activity Defined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micromole of denatured BSA and RNase B in 1 minute at 37°C, pH 7.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster).
Storage Store enzyme at 4°C.