PNGase F
Peptide-N-Glycosidase F (PNGase F) waa habka enzymatic ugu waxtarka badan ee ka saarida ku dhawaad dhammaan oligosaccharides N-linked ee glycoproteins.PNGase F waa amidase, kaas oo kala sooca inta badan GlcNAc gudaha iyo hadhaaga asparagine ee mannose sare, isku-dhafka, iyo oligosaccharides adag ee glycoprotein-ku xiran.
Codsiga
Insaymiskani wuxuu faa'iido u leeyahay ka saarista hadhaaga karbohaydrayt ee borotiinnada.
Diyaarinta iyo qeexidda
| Muuqashada | Dareere aan Midab lahayn |
| Nadiifnimada borotiinka | ≥95% (laga bilaabo SDS-PAGE) |
| Hawsha | ≥500,000 U/ml |
| Exoglycosidase | Wax dhaqdhaqaaq ah lama ogaan karo (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H | ND |
| Protease | ND |
Guryaha
| lambarka EC | 3.5.1.52(Ka-soo-noqoshada noolaha) |
| Miisaanka molecular | 35 kDa (SDS-BOGGA) |
| Barta koronto | 8.14 |
| pH ugu fiican | 7.0-8.0 |
| Heerkulka ugu wanaagsan | 65 °C |
| Gaar ahaan substrate | Goynta curaarta glycosidic ee u dhaxaysa GlcNAc iyo hadhaaga asparagine Jaantus.1 |
| Goobaha aqoonsiga | N-linked glycans ilaa ay ku jiraan α1-3 fukose sawirka 2 |
| Dhaqdhaqaaqayaasha | DTT |
| Kahortagga | SDS |
| Heerkulka kaydinta | -25 ~-15 ℃ |
| Dhaqdhaqaaq la'aanta kulaylka | Isku darka falcelinta 20µL oo ka kooban 1µL oo PNGase F ah ayaa lagu damiyay kuleejka 75 °C ilaa 10 daqiiqo. |
Jaantuska 1 Gaar ahaan Substrate-ka ee PNGase F
Jaantuska. 2 fadhiyada aqoonsiga ee PNGase F.
Marka hadhaaga GlcNAc gudaha ay ku xidhan yihiin α1-3 fucose, PNGase F kama saari karo oligosaccharides N-linked ee glycoproteins.Wax ka beddelkani waxa uu ku badan yahay dhirta iyo glycoproteinnada cayayaanka qaarkood.
Cxoogsheegayaal
|
| Qaybaha | Xoog-saarid |
| 1 | PNGase F | 50 µl |
| 2 | 10×Glycoprotein Denatureing Buffer | 1000 µl |
| 3 | 10×GlycoBuffer 2 | 1000 µl |
| 4 | 10% NP-40 | 1000 µl |
Qeexitaanka unugga
Hal unug(U) ayaa lagu qeexaa inay tahay cadadka insaymka loo baahan yahay si looga saaro>95% karbohaydraytka 10 µg ee RNase B ee denataysan 1 saac gudahooda 37°C wadarta mugga falcelinta ee 10 µL.
Xaaladaha falcelinta
1. Ku mil 1-20 µg oo glycoprotein ah oo leh biyo fuudaysan, ku dar 1 µl 10 × Glycoprotein Denaturing Buffer iyo H2O (haddii loo baahdo) si loo sameeyo 10 µl mugga falcelinta.
2.Ku dhaji 100 ° C 10 daqiiqo, ku qabooji baraf.
3.Ku dar 2µl 10×GlycoBuffer 2, 2 µl 10% NP-40 oo qas.
4.Ku dar 1-2 µl PNGase F iyo H2O (haddii loo baahdo) si loo sameeyo 20 µl mugga falcelinta oo isku dar ah.
5.Dareen-celinta 37 ° C 60 daqiiqo.
6.Falanqaynta SDS-PAGE ama falanqaynta HPLC.
