PNGase F.
Peptide-N-Glycosidase F (PNGase F) nuburyo bwiza bwimisemburo ikuraho hafi ya N-ihuza oligosakisaride hafi ya glycoproteine.PNGase F ni amidase, ikomatanya hagati yimbere ya GlcNAc yimbere hamwe nibisigara bya asparagine bya mannose ndende, hybrid, hamwe na oligosakisaride igoye ituruka kuri N-ihuza glycoproteine.
Gusaba
Iyi misemburo ni ingirakamaro mu gukuraho ibisigazwa bya karubone ya poroteyine.
Gutegura no gusobanura
| Kugaragara | Amazi adafite ibara |
| Intungamubiri za poroteyine | ≥95% (kuva kuri SDS-PAGE) |
| Igikorwa | 500.000 U / mL |
| Exoglycosidase | Nta gikorwa na kimwe gishobora kugaragara (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H. | ND |
| Kurinda | ND |
Ibyiza
| Numero ya EC | 3.5.1.52(Recombinant kuva mikorobe) |
| Uburemere bwa molekile | 35 kDa (SDS-PAGE) |
| Ingingo ya Isoelectric | 8. 14 |
| PH nziza | 7.0-8.0 |
| Ubushyuhe bwiza | 65 ° C. |
| Kwerekana umwihariko | Kurandura glycosidique ihuza GlcNAc n'ibisigazwa bya asparagine Igishusho.1 |
| Imbuga zo kumenyekana | N-ihuza glycans keretse irimo α1-3 fucose Igishushanyo 2 |
| Abakangurambaga | DTT |
| Inhibitor | SDS |
| Ubushyuhe bwo kubika | -25 ~ -15 ℃ |
| Gushyushya | Imvange ya 20µL irimo 1µL ya PNGase F idakorwa na incubation kuri 75 ° C muminota 10. |
Igishushanyo 1 Sobanura umwihariko wa PNGase F.
Igishushanyo 2 Kumenyekanisha bicaye kuri PNGase F.
Iyo GlcNAc yimbere isigaye ihujwe na α1-3 fucose, PNGase F ntishobora gukuramo N-ihuza oligosaccharide ya glycoproteine.Ihinduka risanzwe mubihingwa hamwe na glycoproteine zimwe na zimwe.
CIbigize
|
| Ibigize | Kwibanda |
| 1 | PNGase F. | 50 µl |
| 2 | 10 × Glycoprotein Yerekana Buffer | 1000 µl |
| 3 | 10 × GlycoBuffer 2 | 1000 µl |
| 4 | 10% NP-40 | 1000 µl |
Igisobanuro cyibice
Igice kimwe (U) gisobanurwa nkubunini bwa enzyme isabwa kugirango ikureho> 95% ya karubone ya hydrata kuri 10 µg ya RNase B yangiritse mumasaha 1 kuri 37 ° C mubisubizo byuzuye bya 10 µL.
Ibisabwa
1.Gabanya 1-20 µg ya glycoproteine hamwe namazi ya deioniyo, ongeramo 1 µl 10 × Glycoprotein Denaturing Buffer na H2O (nibiba ngombwa) kugirango ukore 10 µl yuzuye.
2.Shyira kuri 100 ° C muminota 10, ukonje kurubura.
3.Ongeramo 2 µl 10 × GlycoBuffer 2, 2 µl 10% NP-40 hanyuma uvange.
4.Ongeramo 1-2 µl PNGase F na H.2O (nibiba ngombwa) gukora 20 µl yuzuye reaction hanyuma ukavanga.
5.Incubate reaction kuri 37 ° C kuminota 60.
6.Isesengura rya SDS-PAGE cyangwa isesengura rya HPLC.
