I-Dithiothreitol (i-DTT) yi-arhente yokunciphisa esetyenziswa ngokuqhelekileyo, eyaziwa ngokuba yi-additive entsha eluhlaza.Yinto encinci ye-molecular organic compound enamaqela amabini e-mercaptan (-SH).Ngenxa yokunciphisa iipropati kunye nokuzinza, i-DTT isetyenziswa ngokubanzi kwi-biochemistry kunye ne-molecular biology experiments.
Indima ephambili ye-DTT kukunciphisa iibhondi ze-disulfide kwiiprotheni kunye nezinye i-biomolecules.Ibhondi ye-disulfide yinxalenye ebalulekileyo yeprotheyini yokugoqa kunye nokuzinza, kodwa phantsi kweemeko ezithile zovavanyo, ezifana nohlalutyo lwe-SDS-PAGE olunokunciphisa, ukuhlanganiswa kweprotheni kunye nokugoqa, kuyimfuneko ukunciphisa ibhondi ye-disulfide kumaqela amabini e-thiol ukutyhila isakhiwo sendawo iprotheni.I-DTT inokusabela kunye neebhondi ze-disulfide ukuze zinciphise kumaqela e-mercaptan, ngaloo ndlela ivula isakhiwo sendawo yeprotheni kwaye yenza kube lula ukuhlalutya kunye nokusebenza.
I-DTT ingasetyenziselwa ukukhusela umsebenzi we-enzyme kunye nokuzinza.Kwezinye i-enzyme-catalyzed reactions, umsebenzi we-enzyme unokuncitshiswa yi-oxidant.I-DTT inokusabela kunye ne-oxidants ukunciphisa kwizinto ezingenabungozi, ngaloo ndlela ikhusela umsebenzi kunye nokuzinza kwe-enzyme.
Xa kuthelekiswa nama-agent okunciphisa amasiko afana ne-β-mercaptoethanol (β-ME), i-DTT ithathwa njenge-agent yokunciphisa ekhuselekileyo kwaye izinzile.Ayizinzile kuphela kwisisombululo samanzi, kodwa igcina iimpawu zayo zokunciphisa phantsi kweqondo lokushisa eliphezulu kunye neemeko ze-acid-base.
Ukusetyenziswa kwe-DTT kulula.Ngokubanzi, i-DTT inyibilika kwisithinteli esifanelekileyo kwaye emva koko yongezwa kwinkqubo yovavanyo.Ugxininiso olufanelekileyo lwe-DTT lufuna ukuqinisekiswa ngokovavanyo oluthile, kwaye lusetyenziswa ngokubanzi kuluhlu lwe-0.1-1mM.Ukugxininiswa okuphantsi kunokunciphisa imiphumo emibi ekukhuleni kweeseli kwaye kunokunciphisa i-cytotoxicity ngenxa yokugqithiswa kweeprotheni ezijoliswe kuyo.Ukugxila okuphezulu kunokubangela umthwalo ogqithisileyo we-cell metabolic, echaphazela ukukhula kweeseli kunye nokusebenza kakuhle kokubonakalisa.
Indlela yokumisela ugxininiso olufanelekileyo lunokuvavanya inqanaba lokuvakalisa iprotheni ekujoliswe kuyo ngokuqhuba iimvavanyo ze-IPTG zokungeniswa kwiindawo ezahlukeneyo.Uvavanyo lwenkcubeko olukwizinga eliphantsi lunokwenziwa kusetyenziswa uluhlu lwe-IPTG yogxininiso (umz. 0.1 mM, 0.5 mM, 1 mM, njl. njl.) kunye nesiphumo sokubonakaliswa kwiimpawu ezahlukeneyo sinokuvavanywa ngokufumanisa inqanaba lokubonisa iprotheni ekujoliswe kuyo (umz. ukufunyanwa kwebala okanye ukubonwa kwe-fluorescence).Ngokweziphumo zovavanyo, ugxininiso olunesiphumo esihle sokubonisa sikhethwe njengogxininiso olufanelekileyo.
Ukongeza, unokubhekisa kuncwadi olufanelekileyo okanye amava ezinye iilabhoratri ukuqonda uluhlu loxinaniso lwe-IPTG olusetyenziswa ngokuqhelekileyo phantsi kweemeko zovavanyo ezifanayo, kwaye emva koko ulungise kwaye ulungelelanise ngokweemfuno zovavanyo.
Kubalulekile ukuqaphela ukuba ugxininiso olufanelekileyo lunokwahluka ngokuxhomekeke kwiinkqubo zokuvakalisa ezahlukeneyo, iiprotheyini ezijoliswe kuzo, kunye neemeko zovavanyo, ngoko ke kungcono ukukhulisa kwimeko-nge-case basis.
Isishwankathelo, i-DTT yinto eqhelekileyo yokunciphisa i-agent engasetyenziselwa ukunciphisa i-disulfide bonds kwiiprotheni kunye nezinye i-biomolecules kunye nokukhusela umsebenzi we-enzyme kunye nokuzinza.Isetyenziswe ngokubanzi kwi-biochemistry kunye novavanyo lwebhayoloji ye-molekyuli.
Ixesha lokuposa: Sep-28-2023