PNGase F cleaves N-linked (asparagine-linked) oligosaccharides from glycoproteins. The enzyme deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. Denaturation increases the rate of cleavage. Most native proteins can still be completely N-deglycosylated but incubation time must be increased. The enzyme will remain fully active under reaction conditions (37°C) for at least 96 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.

There are a number of alternative enzymes which can be used to remove N-glycans, most especially the Endo F family of enzymes and Endo H. These enzymes cleave between the two N-acetylglucosamine residues in the core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. This leaves a charged sugar which can assist in keeping proteins in solution that precipitate after deglycosylation with PNGase F which removes the oligosaccharide intact. Endo F1 cleaves high mannose and some hybrid type N-glycans. Endo F2 will removes biantennary and high mannose (at a 40X reduced rate). Endo F3 releases of triantennarry and fucosylated biantennary N-glycans. Endo H removes hybrid or high mannose glycans.

Source: Elizabethkingia miricola (was Chryseobacterium meningosepticum)

EC: 3.5.1.52
Alternative Names: PNGase F, Peptide N Glycosidase F, N-Glycosidase, N-Glycanase

Contents E-PNG01:
Ludger PNGase F - Kit contents
PNGase F in 20 mM Tris-HCl, pH 7.5 - 60µL
5x Reaction Buffer 7.5 – 250 mM sodium phosphate, pH 7.5
Denaturation Solution – 2% SDS, 1 M Beta-mercaptoethanol
Triton X-100 – 15% solution

Contents E-PNG01-200 (enzyme only):
PNGase F in 20 mM Tris-HCl, pH 7.5 - 200µL

Specific Activity: >25 U/mg

Activity: 5 U/mL

Molecular weight: 36,000 daltons

pH range: 6-10, optimum 7.5

Protocol:
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 Reaction Buffer 7.5 and 2.5 µL of Denaturation Solution. Heat at 100°C for 5 minutes.
3. Cool. Add 2.5 µL of Triton X-100 and mix.
4. Add 2.0 µL of enzyme to the reaction. Incubate 3 hours at 37°C.

Specificity: 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 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.