PNGase F
Peptide-N-Glycosidase F(PNGase F) ndiyo njira yabwino kwambiri ya enzymatic yochotsera pafupifupi oligosaccharides onse olumikizidwa ndi N kuchokera ku glycoproteins.PNGase F ndi amidase, yomwe imadutsa pakati pa GlcNAc yambiri yamkati ndi zotsalira za asparagine za mannose apamwamba, osakanizidwa, ndi oligosaccharides ovuta kuchokera ku N-linked glycoproteins.
Kugwiritsa ntchito
Enzyme iyi ndiyothandiza pakuchotsa zotsalira zama carbohydrate ku mapuloteni.
Kukonzekera ndi kulongosola
| Maonekedwe | Mtundu Wamadzimadzi |
| Mapuloteni chiyero | ≥95% (kuchokera ku SDS-PAGE) |
| Zochita | ≥500,000 U/mL |
| Exoglycosidase | Palibe ntchito yomwe ingadziwike (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H | ND |
| Protease | ND |
Katundu
| EC nambala | 3.5.1.52(Recombinant kuchokera ku microorganism) |
| Kulemera kwa maselo | 35 kDa (SDS-PAGE) |
| Isoelectric point | 8. 14 |
| Mulingo wabwino kwambiri wa pH | 7.0-8.0 |
| Kutentha koyenera | 65 °C |
| Kukhazikika kwa gawo lapansi | Kuchotsa zomangira za glycosidic pakati pa GlcNAc ndi zotsalira za asparagine Chithunzi 1 |
| Masamba odziwika | N-olumikizidwa ndi glycans pokhapokha mutakhala ndi α1-3 fucose Chithunzi 2 |
| Zoyambitsa | Mtengo wa DTT |
| Choletsa | SDS |
| Kutentha kosungirako | -25 ~ 15 ℃ |
| Kutentha Kutentha | Kusakaniza kwa 20µL komwe kumakhala ndi 1µL ya PNGase F kumayendetsedwa ndi incubation pa 75 °C kwa mphindi 10. |
Chithunzi cha 1 Kagawo kakang'ono ka PNGase F
Chithunzi cha 2 Recognition sits of PNGase F.
Pamene zotsalira za GlcNAc zamkati zimagwirizanitsidwa ndi α1-3 fucose, PNGase F sangathe kuphwanya oligosaccharides yolumikizidwa ndi N kuchokera ku glycoproteins.Kusintha kumeneku kumakhala kofala muzomera ndi tizilombo tina ta glycoproteins.
Cotsutsa
|
| Zigawo | Kukhazikika |
| 1 | PNGase F | 50 μl |
| 2 | 10 × Glycoprotein Denaturing Buffer | 1000µl |
| 3 | 10 × GlycoBuffer 2 | 1000µl |
| 4 | 10% NP-40 | 1000µl |
Tanthauzo la unit
Chigawo chimodzi (U) chimatanthauzidwa ngati kuchuluka kwa enzyme yofunikira kuchotsa> 95% ya chakudya kuchokera ku 10 µg ya RNase B yopangidwa mu ola la 1 pa 37 ° C mu chiwerengero chonse cha 10 µL.
Zomwe zimachitika
1. Sungunulani 1-20 µg ya glycoprotein ndi madzi opangidwa ndi deionized, onjezerani 1 µl 10×Glycoprotein Denaturing Buffer ndi H2O (ngati kuli kofunikira) kuti mupange voliyumu yonse ya 10 µl.
2.Yakani pa 100 ° C kwa mphindi 10, kuziziritsa pa ayezi.
3.Onjezani 2 µl 10×GlycoBuffer 2, 2 µl 10% NP-40 ndikusakaniza.
4.Onjezani 1-2 µl PNGase F ndi H2O (ngati kuli kofunikira) kuti mupange voliyumu yokwana 20 µl ndikusakaniza.
5.Yambani kuchita pa 37 ° C kwa 60 min.
6.Kusanthula kwa SDS-PAGE kapena kusanthula kwa HPLC.
