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Recombinant PNGase F [For Glycan Release] (CAT#: CB-P251-K) Datasheet

Product Type
Kit
Description
PNGase F is suitable for releasing all types (high mannose, hybrid and complex) N-linked glycans from glycoproteins and glycopeptides. PNGaseF does not remove the α(1-3) linkages commonly found on plant glycoproteins The core fucose is an oligosaccharide.
Recombinant PNGaseF was isolated from the E. coli strain containing the Elizabethkingia miricola gene clone. Compared with the natural enzyme (CB-P249-K), there is no detectable difference in the activity or specific activity of the recombinant enzyme.

PNGase F cleaves asparagine-linked (N-linked) oligosaccharides from glycoproteins. PNGase F deaminates asparagine to aspartic acid, leaving oligosaccharides intact. Denaturation can increase the cutting rate up to 100 times. Most natural proteins can still be completely N-deglycosylated, but the incubation time must be increased. PNGase F will remain active for at least 72 hours under incubation conditions. PNGase F will not remove the α(1,3)-linked core fucose oligosaccharides commonly found on plant glycoproteins; for this, please use peptide N-glycosidase A.
Features
PNGase F Source: recombinant PNGase F gene from Elizabethkingia miricola in E. coli
EC: 3.5.1.52
Alternative Names: PNGase F, Peptide N Glycosidase F, N-Glycosidase, N-Glycanase
Specific Activity:>25 U/mg
Activity: 5 U/mL
Molecular weight: 36,000 daltons
pH range for PNGaseF: 6-10, optimum 7.5
Size
0.3 U / 60 µL (Sufficient for up to 60 reactions.)
Sensitivity
Defined 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).
Sample
Glycoproteins and glycopeptides containing N-linked glycans
Species Reactivity
Specificity: 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 α(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A.
Components
60 µL aliquot of recombinant 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
Handling Advice
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 PNGaseF Triton X-100 and mix.
4. Add 2.0 µL of PNGaseF to the reaction. Incubate 3 hours at 37°C.
Storage
Store enzyme at 4°C.
Shelf Life
12 months
Research Use
For research use only. Not for human or drug use

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