PNGase F
I-Peptide-N-Glycosidase F (PNGase F) yeyona ndlela isebenzayo ye-enzymatic yokususa phantse yonke i-oligosaccharides e-N-linked kwi-glycoproteins.I-PNGase F i-amidase, eqhekeza phakathi kweyona ndawo ingaphakathi ye-GlcNAc kunye neentsalela ze-asparagine eziphezulu ze-mannose, i-hybrid, kunye ne-oligosaccharides eyinkimbinkimbi evela kwi-N-linked glycoproteins.
Isicelo
Le enzyme iluncedo ekususeni iintsalela zecarbohydrate kwiiproteni.
Ukulungiselela kunye neenkcukacha
| Imbonakalo | Ulwelo olungenambala |
| Ukucoceka kweeprotheni | ≥95% (ukusuka kwi-SDS-PAGE) |
| Umsebenzi | ≥500,000 U/mL |
| Exoglycosidase | Akukho msebenzi unokufunyanwa (ND) |
| I-Endoglycosidase F1 | ND |
| I-Endoglycosidase F2 | ND |
| I-Endoglycosidase F3 | ND |
| I-Endoglycosidase H | ND |
| Iprotease | ND |
Iipropati
| Inombolo yeEC | 3.5.1.52(I-Recombinant evela kwi-microorganism) |
| Ubunzima bemolekyuli | 35 kDa (SDS-PAGE) |
| Indawo ye-isoelectric | 8. 14 |
| Eyona pH | 7.0-8.0 |
| Ubushushu obuphezulu | 65 °C |
| Ukucaciswa kwesubstrate | Ukucoca amabhondi e-glycosidic phakathi kwe-GlcNAc kunye neentsalela ze-asparagine Fig.1 |
| Iindawo zokuqaphela | I-N-linked glycans ngaphandle kokuba iqulethe i-α1-3 fucose Umfanekiso we-2 |
| Izivuseleli | DTT |
| Isithinteli | SDS |
| Ubushushu bokugcina | -25 ~-15 ℃ |
| Ukushisa Ukungasebenzi | Umxube we-20µL wokusabela oqulethe i-1µL ye-PNGse F uyenziwa ukuba ingasebenzi ngokufukanyelwa kwi-75 °C kangangemizuzu eli-10. |
Isazobe soku-1 esicacileyo seSubstrate ye-PNGase F
Umzobo 2 Uqwalaselo luhlala lwePNGase F.
Xa ii-residues ze-GlcNAc zangaphakathi zidibaniswe ne-α1-3 fucose, i-PNGase F ayikwazi ukuqhawula i-oligosaccharides ye-N-linked kwi-glycoproteins.Olu hlengahlengiso luqhelekileyo kwizityalo kunye nezinye izinambuzane ze-glycoprotein.
Cabachasi
|
| Amacandelo | Ukugxininisa |
| 1 | PNGase F | 50µl |
| 2 | 10 × Glycoprotein Denaturing Buffer | 1000µl |
| 3 | 10×GlycoBuffer 2 | 1000µl |
| 4 | 10% NP-40 | 1000µl |
Inkcazo yeyunithi
Iyunithi enye (U) ichazwa njengobungakanani be-enzyme efunekayo ukususa > i-95% ye-carbohydrate ukusuka kwi-10 µg ye-denatured RNase B ngeyure ye-1 kwi-37 ° C kumthamo opheleleyo wokusabela we-10 µL.
Iimeko zokusabela
1.Nyibilikisa i-1-20 µg ye-glycoprotein ngamanzi adiyoniyoni, yongeza i-1 µl 10×I-Glycoprotein i-Denaturing Buffer kunye ne-H2O (ukuba kuyimfuneko) ukwenza i-10 µl yevolumu yokusabela iyonke.
2.Ufukame kwi-100 ° C ngemizuzu eyi-10, uyipholise kwiqhwa.
3.Yongeza i-2 µl 10×GlycoBuffer 2, 2 µl 10% NP-40 kwaye udibanise.
4.Yongeza i-1-2 µl PNGase F kunye ne-H2O (ukuba kukho imfuneko) ukwenza i-20 µl iyonke ivolumu yokusabela kunye nokuxuba.
5.I-Incubate reaction kwi-37 ° C kwi-60 min.
6.Uhlalutyo lwe-SDS-PAGE okanye uhlalutyo lwe-HPLC.
