PNGase F
Peptide-N-Glycosidase F (PNGase F) jẹ ọna enzymatiki ti o munadoko julọ fun yiyọ gbogbo awọn oligosaccharides ti o ni asopọ N-lati awọn glycoproteins.PNGase F jẹ amidase, eyiti o pin laarin pupọ julọ GlcNAc ati awọn iṣẹku asparagine ti mannose giga, arabara, ati oligosaccharides eka lati awọn glycoproteins N-isopọ.
Ohun elo
Enzymu yii wulo fun yiyọkuro awọn iṣẹku carbohydrate lati awọn ọlọjẹ.
Igbaradi ati sipesifikesonu
| Ifarahan | Omi ti ko ni awọ |
| Amuaradagba ti nw | ≥95% (lati SDS-PAGE) |
| Iṣẹ-ṣiṣe | ≥500,000 U/ml |
| Exoglycosidase | Ko si iṣẹ ṣiṣe ti a le rii (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H | ND |
| Proteate | ND |
Awọn ohun-ini
| EC nọmba | 3.5.1.52(Atunpọ lati microorganism) |
| Ìwúwo molikula | 35 kDa (SDS-iwe) |
| Isoelectric ojuami | 8.14 |
| pH ti o dara julọ | 7.0-8.0 |
| Iwọn otutu to dara julọ | 65 °C |
| Sobusitireti pato | Pipa awọn ifunmọ glycosidic kuro laarin GlcNAc ati awọn iṣẹku asparagine Fig.1 |
| Awọn aaye idanimọ | glycans N-linked ayafi ti o ni α1-3 fucose eeya 2 ninu |
| Awọn aṣiṣẹ | DTT |
| Inhibitor | SDS |
| Iwọn otutu ipamọ | -25 ~-15 ℃ |
| Ooru Inactivation | Adalu ifaseyin 20µL ti o ni 1µL ti PNGase F wa ni aṣiṣẹ nipasẹ abeabo ni 75 °C fun awọn iṣẹju 10. |
Aworan 1 Pataki sobusitireti ti PNGase F
Aworan 2 Awọn ijoko idanimọ ti PNGase F.
Nigbati awọn iṣẹku GlcNAc inu ti sopọ mọ fucose α1-3, PNGase F ko le ya awọn oligosaccharides ti o ni asopọ N lati awọn glycoproteins.Yi iyipada jẹ wọpọ ni awọn eweko ati diẹ ninu awọn glycoproteins kokoro.
Calagbara
|
| Awọn eroja | Ifojusi |
| 1 | PNGase F | 50 µl |
| 2 | 10× Glycoprotein Denatureing saarin | 1000 µl |
| 3 | 10× GlycoBuffer 2 | 1000 µl |
| 4 | 10% NP-40 | 1000 µl |
Itumọ ẹyọkan
Ẹyọ kan (U) jẹ asọye bi iye henensiamu ti o nilo lati yọ> 95% ti carbohydrate kuro lati 10 μg ti denatured RNase B ni wakati 1 ni 37°C ni iwọn ifasẹyin lapapọ ti 10 µL.
Awọn ipo ifaseyin
1.Tu 1-20 µg ti glycoprotein pẹlu omi ti a ti sọ diionized, fi 1µl 10× Glycoprotein Denatuing Buffer ati H2O (ti o ba jẹ dandan) lati ṣe iwọn didun 10 µl lapapọ.
2.Incubate ni 100 ° C fun iṣẹju 10, tutu lori yinyin.
3.Ṣafikun 2µl 10×GlycoBuffer 2, 2 µl 10% NP-40 ki o si dapọ.
4.Ṣafikun 1-2 µl PNGase F ati H2O (ti o ba jẹ dandan) lati ṣe iwọn 20 µl lapapọ ifaseyin ati dapọ.
5.Idahun incubate ni 37°C fun iṣẹju 60.
6.Fun itupalẹ SDS-PAGE tabi itupalẹ HPLC.
