PNGase F
Peptide-N-Glycosidase F (PNGase F) yog txoj hauv kev zoo tshaj plaws rau kev tshem tawm yuav luag txhua N-txuas oligosaccharides los ntawm glycoproteins.PNGase F yog amidase, uas cleaves ntawm sab hauv feem ntau GlcNAc thiab asparagine residues ntawm siab mannose, hybrid, thiab complex oligosaccharides los ntawm N-txuas glycoproteins.
Daim ntawv thov
Cov enzyme no muaj txiaj ntsig zoo rau kev tshem tawm cov carbohydrate seem ntawm cov protein.
Kev npaj thiab specification
| Qhov tshwm sim | Xim tsis muaj kua |
| Protein purity | ≥95% (los ntawm SDS-PAGE) |
| Kev ua si | ≥500,000 U / ml |
| Exoglycosidase | Tsis muaj kev ua haujlwm yuav raug kuaj pom (ND) |
| Endoglycosidase F1 | ND |
| Endoglycosidase F2 | ND |
| Endoglycosidase F3 | ND |
| Endoglycosidase H | ND |
| Protease | ND |
Cov khoom
| EC tus lej | 3.5.1.52 ib(Recombinant los ntawm microorganism) |
| Molecular hnyav | 35 kDa (SDS-PAGE) |
| Isoelectric point | 8.14 ib |
| Qhov zoo tshaj pH | 7.0-8.0 Nws |
| Qhov kub zoo tshaj | 65 ° C |
| Substrate specification | Cleaving glycosidic bonds ntawm GlcNAc thiab asparagine residues Fig.1 |
| Cov chaw paub | N-txuas glycans tshwj tsis yog muaj α1-3 fucose Daim duab 2 |
| Activators | DTT |
| Inhibitor | SDS |
| Cia kub | -25 ~ -15 ℃ |
| Kub Inactivation | Ib qho 20µL cov tshuaj tiv thaiv sib xyaw uas muaj 1µL ntawm PNGase F yog inactivated los ntawm incubation ntawm 75 ° C rau 10 feeb. |
Daim duab 1 Substrate specification ntawm PNGase F
Fig. 2 Kev lees paub zaum ntawm PNGase F.
Thaum GlcNAc cov seem txuas nrog α1-3 fucose, PNGase F tsis tuaj yeem tshem N-txuas oligosaccharides los ntawm glycoproteins.Qhov kev hloov kho no muaj nyob hauv cov nroj tsuag thiab qee cov kab glycoproteins.
Ccov omponents
|
| Cheebtsam | Nco ntsoov |
| 1 | PNGase F | 50 l;ua |
| 2 | 10 × Glycoprotein Denaturing Buffer | 1000 µl |
| 3 | 10 × GlycoBuffer 2 | 1000 µl |
| 4 | 10% NP-40 | 1000 µl |
Unit txhais
Ib chav tsev (U) txhais tau tias yog cov enzyme uas yuav tsum tau tshem tawm> 95% ntawm cov carbohydrate los ntawm 10 µg ntawm denatured RNase B hauv 1 teev ntawm 37 ° C hauv qhov ntim tag nrho ntawm 10 µL.
Cov xwm txheej tshwm sim
1.Dissolve 1-20 µg ntawm glycoprotein nrog dej deionized, ntxiv 1 µl 10 × Glycoprotein Denaturing Buffer thiab H2O (yog tias tsim nyog) ua kom 10 µl tag nrho cov tshuaj tiv thaiv ntim.
2.Ci hauv qhov cub ntawm 100 ° C rau 10 feeb, txias rau hauv dej khov.
3.Ntxiv 2 µl 10 × GlycoBuffer 2, 2 µl 10% NP-40 thiab sib tov.
4.Ntxiv 1-2 µl PNGase F thiab H2O (yog tias tsim nyog) ua kom 20 µl tag nrho cov tshuaj tiv thaiv ntim thiab sib tov.
5.Incubate cov tshuaj tiv thaiv ntawm 37 ° C rau 60 min.
6.Rau SDS-PAGE tsom xam lossis HPLC tsom xam.
