Active Native Elizabethkingia miricola PNGase F

• Cat.No.: PNGase F-011E
• Product Overview: Native Elizabethkingia miricola PNGase F

Specification

• Species: Elizabethkingia miricola
• Description: 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 centigrade) 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. PNGase F cleaves N-linked (asparagine-linked) oligosaccharides from glycoproteins. The enzyme deaminates asparagine to aspartic acid, leaving the oligosaccharides intact. 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. Denaturation increases the rate of cleavage. Most native proteins can still be completely N-deglycosylated but incubation time may need to be increased. The enzyme will remain fully active under reaction conditions (37 centigrade) for at least 96 hours. 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.
• Form: Sterile-filtered solution
• EC: 3.5.1.52
• 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 100×. 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.
• Contents: PNGase F in 20 mM Tris-HCl, pH 7.5-60μL 5× Reaction Buffer 7.5-250 mM sodium phosphate, pH 7.5 Denaturation Solution-2% SDS, 1 M Beta-mercaptoethanol Triton X-100-15% solution
• Bio-activity: Activity: 5 U/mL Specific Activity: > 25 U/mg 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 centigrade, pH 7.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster).
• Molecular Mass: 36 kDa
• 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 5× Reaction Buffer 7.5 and 2.5 μL of Denaturation Solution. Heat at 100 centigrade 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 centigrade.
• Unit Definition: One unit of PNGase F activity is 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 centigrade, pH 7.5. Cleavage is monitored by SDS-PAGE (cleaved RNase B migrates faster).
• Purity: PNGase F is tested for contaminating protease as follows: 10 μg of denatured BSA is incubated at 37 centigrade for 24 hours with 2 μL of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation.
• Applications: PNGase F is suitable for the release of all types (high-mannose, hybrid and complex) N-linked glycans from glycoproteins and glycopeptides. PNGase F will not remove oligosaccharides containing α(1-3) linked core fucose commonly found on plant glycoproteins.
• Stability: Several days exposure to ambient temperatures will not reduce activity. Stable at least 24 months when stored properly.
• Storage: Store enzyme at 4 centigrade.
• Storage Buffer: The enzyme is provided as a sterile-filtered solution in 20 mM Tris-HCl, pH 7.5

Gene Information

• Synonyms: PNGase F; Peptide N Glycosidase F; N-Glycosidase; N-Glycanase; png
• UniProt ID: Q9XBM8