PNGase F
ʻO Peptide-N-Glycosidase F(PNGase F) ka hana enzymatic maikaʻi loa no ka wehe ʻana i nā oligosaccharides pili N-pili mai nā glycoproteins.ʻO PNGase F kahi amidase, e hoʻokaʻawale ana ma waena o ka hapa nui o GlcNAc a me nā koena asparagine o ka mannose kiʻekiʻe, hybrid, a me nā oligosaccharides paʻakikī mai nā glycoproteins pili N.
Palapala noi
He mea pono kēia enzyme no ka wehe ʻana i nā koena carbohydrate mai nā protein.
Hoʻomākaukau a me nā kikoʻī
| Ka nana aku | Wai kala ʻole |
| ʻO ka maʻemaʻe protein | ≥95% (mai SDS-PAGE) |
| Ka hana | ≥500,000 U/mL |
| Exoglycosidase | ʻAʻohe hana i ʻike ʻia (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H | ND |
| Protease | ND |
Waiwai
| Helu EC | 3.5.1.52(Hoʻohui hou mai nā microorganism) |
| Kaumaha molekula | 35 kDa (SDS-PAGE) |
| kiko isoelectric | 8. 14 |
| pH maikaʻi loa | 7.0-8.0 |
| ʻO ka wela kūpono | 65 °C |
| ʻAno kikoʻī o ka substrate | Ke wehe nei i nā mea paʻa glycosidic ma waena o GlcNAc a me nā koena asparagine Fig.1 |
| Nā kahua ʻike | N-linked glycans koe ke loaʻa ka α1-3 fucose Fig. 2 |
| Nā mea hoʻoikaika | DTT |
| Kāohi | SDS |
| Mahana mālama | -25 ~-15 ℃ |
| ʻAʻohe wela | ʻAʻole hoʻohana ʻia ka hui ʻana o 20µL i loko o 1µL o PNGase F ma ka hoʻoulu ʻana ma 75 °C no 10 mau minuke. |
Fig
Fig. 2 Noho ʻike ʻia o PNGase F.
Ke hoʻopili ʻia nā koena GlcNAc kūloko i ka α1-3 fucose, ʻaʻole hiki i PNGase F ke hoʻokaʻawale i nā oligosaccharides pili i N mai nā glycoproteins.He mea maʻamau kēia hoʻololi i nā mea kanu a me kekahi glycoproteins insect.
Cnā mea hoʻokūkū
|
| Nā ʻāpana | Hoʻopaʻa |
| 1 | PNGase F | 50 µl |
| 2 | 10×Glycoprotein Denaturing Buffer | 1000 µl |
| 3 | 10×GlycoBuffer 2 | 1000 µl |
| 4 | 10% NP-40 | 1000 µl |
Wehewehe ʻāpana
Hoʻokahi ʻāpana (U) i wehewehe ʻia ʻo ka nui o ka enzyme e pono ai e wehe i>95% o ka carbohydrate mai 10 µg o RNase B denatured i 1 hola ma 37°C i ka nui o ka nui o ka hopena o 10 µL.
Kūlana pane
1. E hoʻoheheʻe i 1-20 µg o glycoprotein me ka wai deionized, e hoʻohui i 1 µl 10×Glycoprotein Denaturing Buffer a me H2O (inā pono) e hana i ka 10 µl ka nui o ka hopena.
2.E hoʻomoʻa i ka 100 ° C no 10 min, e hoʻomaha i ka hau.
3.Hoʻohui i 2 µl 10×GlycoBuffer 2, 2 µl 10% NP-40 a hui pū.
4.Hoʻohui i ka 1-2 µl PNGase F a me H2ʻO (inā pono) e hana i ka 20 µl ka nui o ka hopena a hui pū.
5.Hoʻopili i ka hopena ma 37 ° C no 60 min.
6.No ka nānā ʻana SDS-PAGE a i ʻole ka nānā ʻana HPLC.
