Active Recombinant Elizabethkingia miricola PNGase F

• Cat.No.: PNGase F-014E
• Product Overview: Recombinant PNGase F gene from Elizabethkingia miricola expressed in E. coli

Specification

• Species: Elizabethkingia miricola
• Source: E. coli
• Tag: Non
• Description: 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. 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 α(1,3)-linked core fucose commonly found on plant glycoproteins; for this purpose, use peptide N-glycosidase A.
• 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: 60 μL aliquot of recombinant PNGase F (0.3 U) in 20 mM Tris-HCl, pH 7.5 5× 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
• 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× PNGase F Reaction Buffer 7.5 and 2.5 μL of PNGase F Denaturation Solution. Heat at 100 centigrade 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 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. The production host strain has been extensively tested and does not produce any detectable glycosidases.
• Stability: Several days exposure to ambient temperatures will not reduce activity. Stable at least 12 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