Catalog No.
YXX04901
Description
Peptide -N-Glycosidase F, also known as PNGase F, is an amidase that cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins
Biological activity
One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 10 μg of denatured RNase B in 1 hour at 37°C in a total reaction volume of 10 μl.
Expression system
E. coli
Species
Elizabethkingia miricola
Protein length
PNGase F is cloned from Elizabethkingia miricola and expressed in E.coli.
Predicted molecular weight
37.08 kDa
Nature
Recombinant
Target
PNGase F
Concentration
80,000 units/ml
Endotoxin level
Please contact with the lab for this information.
Purity
>95% as determined by SDS-PAGE.
Applications
Removal of high mannose N-glycans from glycoproteins
Form
Liquid
Storage buffer
20mM Tris-HCl, pH 7.5, 50mM NaCl, 5mM EDTA, 50% glycerol
Experimental Procedure
Standard Operating Procedure
Denaturing Reaction Conditions:
1. Combine 1-20 µg of glycoprotein, 1 µl of Glycoprotein Denaturing Buffer (10X) and H2O (if necessary) to make a 10 µl total reaction volume.
2. Denature glycoprotein by heating reaction at 100°C for 10 minutes.
3. Chill denatured glycoprotein on ice and centrifuge 10 seconds.
4. Make a total reaction volume of 20 µl by adding 2 µl GlycoBuffer 2 (10X), 2 µl 10% NP-40 and 6 µl H2O.
5. Add 1 µl PNGase F, mix gently.
6. Incubate reaction at 37°C for 1 hour.
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.
Note: To deglycosylate different glycoprotein, longer incubation time may be required.
Non-Denaturing Reaction Conditions:
2.Add 2-5 µl PNGase F, mix gently.
3.Incubate reaction at 37°C for 4 - 24 hours.
Note: To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.
1X Glycoprotein Denaturing Buffer
0.5% SDS
40 mM DTT
1X NP-40
1% NP-40 in MilliQ-H2O
1X GlycoBuffer 2
20 mM Tris,PH 7.5
Shipping
In general, proteins are provided as lyophilized powder/frozen liquid. They are shipped out with dry ice/blue ice unless customers require otherwise.
Stability and Storage
Use a manual defrost freezer and avoid repeated freeze thaw cycles.Store at 2 to 8 °C for one week .Store at -20 to -80 °C for twelve months from the date of receipt.
For research use only. Not for human or drug use.
