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 (E-PNG01).

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.

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

Contents:
Ludger recombinant PNGase F - Kit contents
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

Specific Activity: >25 U/mg

Activity: 5 U/mL

Molecular weight: 36,000 daltons

pH range for PNGaseF: 6-10, optimum 7.5

(Protocol) PNGaseF 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 PNGaseF Triton X-100 and mix.
4. Add 2.0 µL of PNGaseF to the reaction. Incubate 3 hours at 37°C.

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.

Specific Activity: 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).

Storage: Store enzyme at 4°C.