PNGase F
Peptide-N-Glycosidase F(PNGase F) ndiyo inonyanya kushanda enzymatic nzira yekubvisa inenge yese N-yakabatana oligosaccharides kubva glycoprotein.PNGase F is an amidase, iyo inotsemuka pakati pemukati zvakanyanya GlcNAc uye asparagine masara epamusoro mannose, hybrid, uye yakaoma oligosaccharides kubva kuN-yakabatana glycoproteins.
Application
Iyi enzyme inobatsira pakubvisa zvisaririra zvecarbohydrate kubva kumapuroteni.
Kugadzirira uye tsanangudzo
| Chitarisiko | Colorless Liquid |
| Mapuroteni kuchena | ≥95% (kubva kuSDS-PEJI) |
| Chiitiko | ≥500,000 U/mL |
| Exoglycosidase | Hapana chiitiko chingaonekwa (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H | ND |
| Protease | ND |
Properties
| EC nhamba | 3.5.1.52(Recombinant kubva ku microorganism) |
| Molecular uremu | 35 kDa (SDS-PEJI) |
| Isoelectric point | 8. 14 |
| Optimum pH | 7.0-8.0 |
| Optimum tembiricha | 65 °C |
| Substrate chaiyo | Kubvisa zvisungo zveglycosidic pakati peGlcNAc nemasara easparagine Fig.1 |
| Kuzivikanwa nzvimbo | N-yakabatana glycans kunze kwekunge ine α1-3 fucose Mufananidzo 2 |
| Activators | DTT |
| Inhibitor | SDS |
| Kuchengetedza tembiricha | -25 ~-15 ℃ |
| Heat Inactivation | A 20µL reaction musanganiswa ine 1µL yePNGase F inovharwa nekuiswa pa75 °C kwemaminetsi gumi. |
Fig. 1 Substrate chaiyo yePNGase F
Fig. 2 Recognition inogara yePNGase F.
Apo masara emukati eGlcNAc akabatanidzwa ne α1-3 fucose, PNGase F haigoni kutsemura N-linked oligosaccharides kubva kuglycoproteins.Kugadziriswa uku kwakajairika muzvirimwa uye zvimwe zvipembenene glycoproteins.
Cvanopikisa
|
| Zvikamu | Concentration |
| 1 | PNGase F | 50µl |
| 2 | 10 × Glycoprotein Denaturing Buffer | 1000µl |
| 3 | 10×GlycoBuffer 2 | 1000µl |
| 4 | 10% NP-40 | 1000µl |
Tsanangudzo yeyuniti
Imwe unit (U) inotsanangurwa sehuwandu hwe enzyme inodiwa kubvisa > 95% yemakhahydrates kubva ku10 µg ye denatured RNase B muawa 1 pa 37 ° C muhuwandu hwekuita huwandu hwe10 µL.
Maitiro ekuita
1.Dissolve 1-20 µg yeglycoprotein nemvura yakasvibiswa, wedzera 1 µl 10×Glycoprotein Denaturing Buffer uye H2O (kana zvichidiwa) kuti uite 10 µl yakazara reaction volume.
2.Isa pa 100 ° C kwemaminitsi gumi, itonhodze pachando.
3.Wedzera 2 µl 10×GlycoBuffer 2, 2 µl 10% NP-40 uye sanganisa.
4.Wedzera 1-2 µl PNGase F uye H2O (kana zvichidikanwa) kugadzira 20 µl yakazara reaction vhoriyamu uye sanganisa.
5.Incubate reaction pa 37°C for 60 min.
6.Yekuongorora kweSDS-PAGE kana HPLC kuongorora.
