PNGase F
Peptide-N-Glycosidase F (PNGase F) bụ usoro enzymatic kachasị dị irè maka iwepu ihe fọrọ nke nta ka ọ bụrụ oligosaccharides niile N-ejikọta na glycoproteins.PNGase F bụ amidase, nke na-ekewa n'etiti GlcNAc kachasị dị n'ime na nsị asparagine nke nnukwu mannose, ngwakọ, na mgbagwoju oligosaccharides sitere na glycoproteins N-njikọ.
Ngwa
Enzyme a bara uru maka iwepụ ihe fọdụrụ nke carbohydrate na protein.
Nkwadebe na nkọwapụta
| Ọdịdị | Mmiri na-enweghị agba |
| Protein dị ọcha | ≥95% (sitere na SDS-PAGE) |
| Ihe omume | ≥500,000 U/ml |
| Exoglycosidase | Enweghị ike ịhụ ọrụ ọ bụla (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H | ND |
| Mgbochi | ND |
Njirimara
| Nọmba EC | 3.5.1.52(Mgbakwunye site na microorganism) |
| Ibu molekụla | 35kDa (SDS-PAGE) |
| Isi ihe nke isoelectric | 8.14 |
| pH kacha mma | 7.0-8.0 |
| Okpomọkụ kacha mma | 65 Celsius C |
| Nkọwapụta mkpụrụ | Iwepu njikọ glycosidic n'etiti GlcNAc na ihe fọdụrụ asparagine Fig.1 |
| Saịtị nnabata | N-njikọ glycans belụsọ nwere α1-3 fucose fig. 2 |
| Ndị na-eme ihe | DTT |
| Ihe mgbochi | SDS |
| Ọnọdụ nchekwa | -25 ~-15 ℃ |
| Ọkụ anaghị arụ ọrụ | Ngwakọta mmeghachi omume 20µL nwere 1µL nke PNGase F na-emenyụ ya site na incubation na 75 Celsius C maka nkeji 10. |
Ihe oyiyi 1 nkọwapụta mkpụrụ nke PNGase F
Fig. 2 Ebe nnabata nke PNGase F.
Mgbe ejikọrọ ihe fọdụrụ n'ime GlcNAc na α1-3 fucose, PNGase F enweghị ike ịwepu oligosaccharides N-njikọ na glycoproteins.Mgbanwe a na-adịkarị na osisi na ụfọdụ glycoproteins ụmụ ahụhụ.
Cndị ọchịchị
|
| Ngwa | Ntinye uche |
| 1 | PNGase F | 50 µl |
| 2 | 10 × Glycoprotein Denaturing Buffer | 1000 µl |
| 3 | 10×GlycoBuffer 2 | 1000 µl |
| 4 | 10% NP-40 | 1000 µl |
Nkọwa nkeji
A kọwapụtara otu nkeji (U) dị ka ọnụọgụ enzyme achọrọ iji wepụ> 95% nke carbohydrate site na 10 μg nke RNase B nke denaturered n'ime elekere 1 na 37 Celsius na mkpokọta mmeghachi omume nke 10 µL.
Ọnọdụ mmeghachi omume
1.Dissolve 1-20 µg nke glycoprotein na mmiri deionized, tinye 1 µl 10 × Glycoprotein Denaturing Buffer na H2O (ọ bụrụ na ọ dị mkpa) iji mee 10 µl mkpokọta mmeghachi omume.
2.Tinye na 100 Celsius C maka nkeji 10, kpoo ya na ice.
3.Tinye 2µl 10×GlycoBuffer 2, 2 µl 10% NP-40 na mix.
4.Tinye 1-2 µl PNGase F na H2O (ọ bụrụ na ọ dị mkpa) iji mee mkpokọta mmeghachi omume 20 µl na mix.
5.Tinye mmeghachi omume na 37 ° C maka nkeji 60.
6.Maka nyocha SDS-PAGE ma ọ bụ nyocha HPLC.
