Active Recombinant Elizabethkingia miricola PNGase F

Cat.No. : PNGase F-014E
Product Overview : Recombinant PNGase F gene from Elizabethkingia miricola expressed in E. coli
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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
Synonyms PNGase F; Peptide N Glycosidase F; N-Glycosidase; N-Glycanase

Not For Human Consumption!

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