Rnase A
I-Ribonuclease A (RNaseA) iyi-polypeptide enomucu owodwa equkethe amabhondi angu-4 e-disulfide enesisindo samangqamuzana esingaba ngu-13.7 kDa.I-RNase A iyi-endoribonuclease eyehlisa ngokukhethekile i-RNA enomucu owodwa ezinsaleni zika-C no-U.Ngokuqondile, i-cleavage ibona isibopho se-phosphodiester esakhiwe yi-5'-ribose ye-nucleotide neqembu le-phosphate ku-3'-ribose ye-nucleotide eseduze ye-pyrimidine, ukuze i-2, 3'-Cyclic phosphates i-hydrolyzed ku-3 ehambisanayo. ama-nucleoside phosphates (isb. i-pG-pG-pC-pA-pG ihlukaniswa yi-RNase A ukuze kukhiqizwe i-pG-pG-pCp kanye ne-A-PG).I-RNase A iyona esebenza kakhulu ekuhlukaniseni i-RNA enomucu owodwa.Ukugxilisa ingqondo okunconyiwe ngu-1-100μG/mL, kuhambisana nezinhlelo zokusabela ezihlukahlukene.Ukugxiliswa kukasawoti okuphansi (0-100 mM NaCl) kungasetshenziswa ukusika i-RNA enomucu owodwa, i-RNA enemicu ekabili, namaketango e-RNA akhiwe yi-RNA-DNA hybridization.Nokho, ekugxilweni kukasawoti omningi (≥0.3 M), i-RNase A ihlukanisa kuphela i-RNA enomucu owodwa.
I-RNase A isetshenziswa kakhulu ukususa i-RNA ngesikhathi sokulungiswa kwe-plasmid DNA noma i-genomic DNA. Ukuthi i-DNase iyasebenza noma cha phakathi nenqubo yokulungiselela kungathinta kalula ukusabela.Indlela yendabuko yokubilisa emanzini okugeza ingasetshenziswa ukwenza umsebenzi we-DNase ungasebenzi.Lo mkhiqizo awunayo i-DNase ne-protease, futhi awudingi ukwelashwa ngokushisa ngaphambi kokusetshenziswa.Ngaphezu kwalokho, lo mkhiqizo ungasetshenziswa futhi ekuhlolweni kwebhayoloji yamangqamuzana njengokuhlaziywa kokuvikelwa kwe-RNase nokuhlaziywa kokulandelana kwe-RNA.
Izimo Zokugcina
Umkhiqizo ungagcinwa ku -25～-15℃, usebenze iminyaka emi-2.
Iziyalezo
Lena enye yezindlela ezivamile zokulungiselela isisombululo se-RNase A.Ingabuye ilungiswe nguezinye izindlela ngokwezindlela zendabuko elabhorethri noma izincwadi eziyinkomba (njengeukuncibilika ngokuqondile ku-10 mM Tris-HCl, pH 7.5 noma isixazululo se-Tris-NaCl)
1. Sebenzisa i-sodium acetate engu-10 mM (pH 5.2) ukulungisa u-10 mg/mL wesisombululo sesitoreji se-RNase A
2. Ukushisa ku-100 ℃ imizuzu engu-15
3. Kuphole ekamelweni lokushisa, engeza ivolumu engu-1/10 ye-1 M Tris-HCl (pH 7.4), lungisa i-pH yayo ibe ngu-7.4 (okweisibonelo, engeza u-500 ml wesisombululo sesitoreji esingu-10g/ml RNase 1M Tris-HCL, PH7.4)
4. Ipakishwe kancane kokuthi -20℃ ukuze igcinwe eqandisiwe, ekwazi ukuzinza iminyaka emi-2.
[Amanothi]: Uma ubilisa isisombululo se-RNaseA ngaphansi kwezimo ezingathathi hlangothi, imvula ye-RNase izokwakhiwa;Yibilise nge-pH ephansi, futhi uma kunemvula, ingabonwa, okungenzeka kubangelwe ukuba khona kokungcola kwamaprotheni.Uma inzika itholakala ngemva kokubilisa, ukungcola kungasuswa nge-high-speed centrifugation (13000rpm), bese kupakishwa kancane ukuze kugcinwe iqhwa.
Ulwazi lomkhiqizo
| Omqondofana | I-Ribonuclease I;i-ribonuclease ye-pancreatic;I-Ribonuclease 3'-i-pyrimidnooligonucleotidohydrolase;Rnase A;Endoribonulcease I |
| Inombolo ye-CAS. | 9001-99-4 |
| Ukubukeka | White lyophilized powder |
| Isisindo samangqamuzana | ~ 13.7kDa (ukulandelana kwe-amino acid) |
| Ph Inani | 7.6 (ibanga lomsebenzi 6-10) |
| Izinga Lokushisa Elifanelekile | 60℃ (Ibanga lomsebenzi 15-70℃) |
| I-ejenti evulayo | Na2+.K+ |
| Inhibitor | I-Rnase Inhibitor |
| Indlela yokungasebenzi | Ayikwazi ukwenziwa isebenze ngokushisisa, kuphakanyiswa ukuthi kusetshenziswe ikholomu ye-centrifuge |
| Umsuka | Inkomo |
| Ukuncibilika | Incibilika emanzini (10mg/ml) |
| Ukulahlekelwa kokomile | ≤5.0% |
| Umsebenzi wama-enzyme | ≥60 amayunithi e-Kunitz/mg |
| Iphuzu le-isoelectric | 9.6 |
Amanothi
Ukuze uphephe kanye nempilo yakho, sicela ugqoke amajazi elebhu namagilavu alahlwayo ukuze usebenze.
