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 |