PNGase F
Peptide-N-Glycosidase F(PNGase F) ke mokhoa o sebetsang ka ho fetisisa oa enzymatic oa ho tlosa hoo e batlang e le li-oligosaccharides tsohle tse amanang le N ho tloha ho glycoprotein.PNGase F ke amidase, e phunyeletsang pakeng tsa GlcNAc e ngata ka hare le masala a asparagine a mannose e phahameng, lebasetere le li-oligosaccharides tse rarahaneng ho tloha ho N-linked glycoprotein.
Kopo
Enzyme ena e thusa ho tlosa masala a carbohydrate ho tsoa liprotheineng.
Tokisetso le tlhaloso
| Ponahalo | Mokelikeli o se nang Mebala |
| Bohloeki ba liprotheine | ≥95% (ho tloha ho SDS-PAGE) |
| Ketsahalo | ≥500,000 U/mL |
| Exoglycosidase | Ha ho ts'ebetso e ka bonoang (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H | ND |
| Protease | ND |
Thepa
| Nomoro ea EC | 3.5.1.52(Recombinant e tsoang ho microorganism) |
| Boima ba molek'hule | 35 kDa (SDS-PAGE) |
| Ntlha ea Isoelectric | 8. 14 |
| Optimum pH | 7.0-8.0 |
| Optimum mocheso | 65 °C |
| Substrate e ikhethang | Ho hlakola maqhama a glycosidic pakeng tsa GlcNAc le masala a asparagine Fig.1 |
| Libaka tsa ho tsebahatsa | N-linked glycans ntle le haeba e na le α1-3 fucose Setšoantšo sa 2 |
| Li-activators | DTT |
| Inhibitor | SDS |
| Mocheso oa polokelo | -25 ~-15 ℃ |
| Ho se sebetse ha mocheso | Motsoako oa karabelo oa 20µL o nang le 1µL ea PNGase F ha o sebetse ka ho kenya 75 °C metsotso e 10. |
Setšoantšo sa 1 Sebopeho sa Substrate sa PNGase F
Setšoantšo sa 2 Recognition sits ea PNGase F.
Ha masala a ka hare a GlcNAc a kopantsoe le α1-3 fucose, PNGase F e ke ke ea arola li-oligosaccharides tsa N-linked ho tloha ho glycoprotein.Phetoho ena e tloaelehile limela le likokoanyana tse ling tsa glycoprotein.
Cbahanyetsi
|
| Likaroloana | Ho tsepamisa maikutlo |
| 1 | PNGase F | 50µl |
| 2 | 10 × Glycoprotein Denaturing Buffer | 1000µl |
| 3 | 10×GlycoBuffer 2 | 1000µl |
| 4 | 10% NP-40 | 1000µl |
Tlhaloso ea yuniti
Yuniti e le 'ngoe (U) e hlalosoa e le palo ea enzyme e hlokahalang ho tlosa > 95% ea lik'habohaedreite ho tloha ho 10 µg ea RNase B e entsoeng ka hora ka hora e le 1 ho 37 ° C ka kakaretso ea karabelo ea 10 µL.
Maemo a ho itšoara
1. Hlakola 1-20 µg ea glycoprotein ka metsi a ntšitsoeng, eketsa 1 µl 10×Glycoprotein Denaturing Buffer le H2O (ha ho hlokahala) ho etsa bophahamo ba 10 µl kakaretso ea karabelo.
2.E kenye ka 100 ° C ka metsotso e 10, e pholile holim'a leqhoa.
3.Kenya 2 µl 10×GlycoBuffer 2, 2 µl 10% NP-40 'me u kopanye.
4.Kenya 1-2 µl PNGase F le H2O (haeba ho hlokahala) ho etsa bophahamo ba molumo oa 20 µl kaofela le ho kopanya.
5.Eketsa karabelo ho 37°C bakeng sa 60 min.
6.Bakeng sa tlhahlobo ea SDS-PAGE kapa tlhahlobo ea HPLC.
