PNGase F*
Peptide-N-Glycosidase F (PNGase F) efficacissima est methodus enzymatica ad tollendum fere omnes N oligosaccharides e glycoproteinis connexis.PNGase F est amidase, quae inter intimiorem GlcNAc et asparaginum residua gravissimae mannosae, hybridis, et oligosaccharidis implicatae ex Glycoprotein N connexis cohaeret.
Applicationem
Hoc enzyme utile est ad residua carbohydratorum tollenda a servo.
Praeparatio et specificatio
| Aspectus | Hyalina Liquid |
| Dapibus pudicitia | ≥95% (ex SDS-PAGE) |
| Actio | ≥500,000 U/mL |
| Exoglycosidase | Nulla actio deprehendi (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H | ND |
| Protease | ND |
Properties
| EC numerus | 3.5.1.52(Recombinante microorganismo) |
| M. pondus | 35 kDa (SDS-PAGE) |
| Isoelectric punctum | 8. 14 |
| optimum pH | 7.0-8.0 |
| Optimum temperatus | 65 °C |
| Substratum proprietatem | Vincula glycosidica inter GlcNAc et asparagines residua inhaerens Fig.1 |
| Recognitio situs | N glycans coniunctum, nisi continet α1-3 fucosae p. 2 |
| Activators | DTT |
| Inhibitor | SDS |
| Repono temperatus | -25 ~-15 |
| Calor Inactivation | A 20µL reactionem commixtio continens 1µL of PNGase F incubatione 75 °C inducitur per 10 minuta. |
P. I Substrate PNGase proprietas F *
Fig.
Cum residua GlcNAc interna cum fucoso α1-3 coniunguntur, PNGase F inhaerere non potest N oligosaccharides e glycoproteinis connexis.Haec modificatio communis est in plantis et glycoproteinis quibusdam insectis.
Componentes
|
| Components | Coniunctis |
| 1 | PNGase F* | 50 µl |
| 2 | 10×Glycoprotein Buffer Denaturing | 1000 µl |
| 3 | 10×GlycoBuffer 2 | 1000 µl |
| 4 | 10% NP-40 | 1000 µl |
Unitas definition
Una unitas(U) definitur ut quantitas enzymi ad removendum >95% carbohydratorum ab 10 µg denaturae RNase B in hora 1 37°C in totali reactione 10 µL.
Reactionem conditionibus
1. Dissolve 1-20 µg glycoproteini cum aqua deionizata, adde 1 µl 10×Glycoprotein Buffer et H2O (si opus est) ut faciat volumen 10 µl totum reactionem.
2.Incubare 100°C pro 10 min, glaciem refrigerare.
3.Adde 2 µl 10×GlycoBuffer 2, 2 µl 10% NP-40 et misce.
4.Adde 1-2 µl PNGase F et H2O (si opus est) ut totum volumen 20 µl reactionem misceat.
5.Incubare reactionem in 37°C pro 60 min.
6.Pro analysi SDS-Paginæ seu analysi HPLC.
