I-Dithiothreitol (DTT) iyi-ejenti yokunciphisa evame ukusetshenziswa, eyaziwa nangokuthi isithasiselo esisha esiluhlaza.Kuyinhlanganisela encane yamangqamuzana enamaqembu amabili e-mercaptan (-SH).Ngenxa yokunciphisa izakhiwo nokuzinza, i-DTT isetshenziswa kakhulu ekuhlolweni kwe-biochemistry kanye ne-molecular biology.
Indima eyinhloko ye-DTT ukunciphisa izibopho ze-disulfide kumaprotheni namanye ama-biomolecules.Isibopho se-disulfide siyingxenye ebalulekile yokugoqa amaprotheni kanye nokuzinza, kodwa ngaphansi kwezimo ezithile zokuhlola, njengokuhlaziywa kwe-SDS-PAGE e-reducible, ukuhlanganiswa kwamaprotheni kanye nokugoqa, kuyadingeka ukunciphisa isibopho se-disulfide emaqenjini amabili e-thiol ukuze kwembule isakhiwo sendawo amaprotheni.I-DTT ingasabela ngezibopho ze-disulfide ukuze zinciphise zibe amaqembu e-mercaptan, ngaleyo ndlela ivule isakhiwo sendawo yeprotheni futhi yenze kube lula ukuyihlaziya nokuyiphatha.
I-DTT ingasetshenziswa futhi ukuvikela umsebenzi we-enzyme nokuzinza.Kwezinye izimo ze-enzyme-catalyzed, umsebenzi we-enzyme ungase uncishiswe yi-oxidant.I-DTT ingasabela nama-oxidants ukuze awehlisele ezintweni ezingenabungozi, ngaleyo ndlela avikele umsebenzi nokuzinza kwe-enzyme.
Uma kuqhathaniswa nama-ejenti okunciphisa endabuko afana ne-β-mercaptoethanol (β-ME), i-DTT ibhekwa njenge-ejenti yokunciphisa ephephile futhi ezinzile.Ayizinzile kuphela kwisisombululo se-aqueous, kodwa futhi igcina izakhiwo zayo zokunciphisa ngaphansi kokushisa okuphezulu kanye nezimo ze-acid-base.
Ukusetshenziswa kwe-DTT kulula.Ngokuvamile, i-DTT incibilika ku-buffer efanele bese yengezwa ohlelweni lokuhlola.Ukugxila okuphelele kwe-DTT kudinga ukunqunywa ngokuvumelana nokuhlolwa okuthile, futhi ngokuvamile kusetshenziswa kububanzi obungu-0.1-1mM.Ukugxila okuphansi kunganciphisa imiphumela emibi ekukhuleni kweseli futhi kunganciphisa i-cytotoxicity ngenxa yokweqisa ngokweqile kwamaprotheni okuhlosiwe.Ukugxila okuphezulu kungase kubangele umthwalo omkhulu we-cell metabolism, kuthinte ukukhula kwamangqamuzana nokusebenza kahle kwenkulumo.
Indlela yokunquma ukugxilisa ingqondo okuphelele kungaba ukuhlola izinga lenkulumo yephrotheni eqondiwe ngokwenza izivivinyo zokungeniswa kwe-IPTG ezindaweni ezihlukene.Ukuhlolwa kwesiko lezinga elincane kungenziwa kusetshenziswa uhla lokugxilisa kwe-IPTG (isb. 0.1 mM, 0.5 mM, 1 mM, njll.) kanye nomthelela wenkulumo ekugxilweni okuhlukene kungahlaziywa ngokuthola izinga lenkulumo yephrotheni eqondiwe (isb. ukubona i-blot noma i-fluorescence).Ngokusho kwemiphumela yokuhlola, ukugxilisa ingqondo okunomphumela omuhle kakhulu wokuveza kukhethwe njengokugxilisa okungcono kakhulu.
Ngaphezu kwalokho, ungaphinda ubhekisele ezincwadini ezifanele noma ulwazi lwamanye amalabhorethri ukuze uqonde ububanzi bokuhlushwa be-IPTG obusetshenziswa kakhulu ngaphansi kwezimo zokuhlola ezifanayo, bese ulungiselela futhi ulungise ngokuya ngezidingo zokuhlola.
Kubalulekile ukuqaphela ukuthi ukugxilisa ingqondo okuhle kungase kwehluke kuye ngamasistimu enkulumo ahlukene, amaprotheni okuqondiwe, nezimo zokuhlola, ngakho-ke kungcono kakhulu ukukhulisa ngokwesisekelo secala ngalinye.
Kafushane, i-DTT iyi-ejenti yokunciphisa evame ukusetshenziswa engasetshenziswa ukunciphisa izibopho ze-disulfide kumaprotheni namanye ama-biomolecules nokuvikela umsebenzi we-enzyme nokuzinza.Isetshenziswe kabanzi ekuhlolweni kwe-biochemistry kanye ne-molecular biology.
Isikhathi sokuthumela: Sep-28-2023