PNGase F
Peptide-N-Glycosidase F (PNGase F) ita ce mafi inganci hanyar enzymatic don cire kusan duk N-linked oligosaccharides daga glycoproteins.PNGase F shine amidase, wanda ke rabe tsakanin mafi yawan GlcNAc na ciki da ragowar asparagine na babban mannose, matasan, da hadaddun oligosaccharides daga glycoproteins masu alaƙa da N.
Aikace-aikace
Wannan enzyme yana da amfani don cire ragowar carbohydrate daga sunadarai.
Shiri da ƙayyadaddun bayanai
| Bayyanar | Ruwa mara launi |
| Tsaftar furotin | ≥95% (daga SDS-PAGE) |
| Ayyuka | ≥500,000 U/ml |
| Exoglycosidase | Ba za a iya gano wani aiki ba (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H | ND |
| Protease | ND |
Kayayyaki
| lambar EC | 3.5.1.52(Recombinant daga microorganism) |
| Nauyin kwayoyin halitta | 35 kDa (SDS-SHAFIN) |
| Isoelectric batu | 8.14 |
| Mafi kyawun pH | 7.0-8.0 |
| Mafi kyawun zafin jiki | 65 °C |
| Ƙayyadaddun Substrate | Yanke haɗin gwiwar glycosidic tsakanin GlcNAc da ragowar asparagine Fig.1 |
| Shafukan ganewa | N-linked glycans sai dai idan ya ƙunshi α1-3 fucose Fig. 2 |
| Masu kunnawa | DTT |
| Mai hanawa | SDS |
| Yanayin ajiya | -25 ~ -15 ℃ |
| Rashin kunna zafi | Cakuda 20µL mai ɗauke da 1µL na PNGase F an kunna shi ta shiryawa a 75 °C na mintuna 10. |
Hoto 1 Ƙayyadaddun Ƙirar PNGase F
Hoto 2 Gane zaune na PNGase F.
Lokacin da ragowar GlcNAc na ciki ke da alaƙa da α1-3 fucose, PNGase F ba zai iya raba N-linked oligosaccharides daga glycoproteins.Wannan gyare-gyare ya zama ruwan dare a cikin tsire-tsire da wasu glycoproteins na kwari.
Cmasu kai hari
|
| Abubuwan da aka gyara | Hankali |
| 1 | PNGase F | 50ml ku |
| 2 | 10× Glycoprotein Denaturing Buffer | 1000 µl |
| 3 | 10×GlycoBuffer 2 | 1000 µl |
| 4 | 10% NP-40 | 1000 µl |
Ma'anar raka'a
An bayyana raka'a ɗaya (U) azaman adadin enzyme da ake buƙata don cire> 95% na carbohydrate daga 10 μg na RNase B a cikin sa'a 1 a 37 ° C a cikin jimlar amsawar 10 µL.
Yanayin amsawa
1.Narke 1-20 µg na glycoprotein tare da ruwa mai lalacewa, ƙara 1 µl 10 × Glycoprotein Denaturing Buffer da H2O (idan ya cancanta) don yin 10 µl jimlar amsawa.
2.Sanya a 100 ° C na minti 10, kwantar da shi a kan kankara.
3.Ƙara 2 µl 10 × GlycoBuffer 2, 2 µl 10% NP-40 kuma gauraya.
4.Ƙara 1-2 µl PNGase F da H2O (idan ya cancanta) don yin 20 µl jimlar ƙarar amsawa da gauraya.
5.Sanya amsa a 37 ° C na minti 60.
6.Don nazarin SDS-PAGE ko bincike na HPLC.
