I-Dithiothreitol (DTT) sisixhobo sokunciphisa esisetyenziswa kakhulu, esaziwa ngokuba sisixhobo esitsha esiluhlaza. Yikhompawundi encinci ye-molecular organic enamaqela amabini e-mercaptan (-SH). Ngenxa yeempawu zayo zokunciphisa kunye nozinzo, i-DTT isetyenziswa kakhulu kwi-biochemistry kunye ne-molecular biology experiments.
Indima ephambili ye-DTT kukunciphisa iibhondi ze-disulfide kwiiproteni nakwezinye ii-biomolecules. Ibhondi ye-disulfide yinxalenye ebalulekileyo yokugoba kunye nokuzinza kweproteni, kodwa phantsi kweemeko ezithile zovavanyo, ezifana nohlalutyo lwe-SDS-PAGE oluncitshiswayo, ukuhlanganiswa kweproteni kunye nokugoba, kuyimfuneko ukunciphisa ibhondi ye-disulfide ukuya kumaqela amabini e-thiol ukuze kutyhilwe isakhiwo sendawo seproteni. I-DTT inokusabela ngeebhondi ze-disulfide ukuze izinciphise zibe ngamaqela e-mercaptan, ngaloo ndlela ivula isakhiwo sendawo seproteni kwaye yenze kube lula ukuyihlalutya nokuyilawula.
I-DTT ingasetyenziselwa ukukhusela umsebenzi wee-enzyme kunye nokuzinza. Kwezinye ii-enzyme-catalyzed reactions, umsebenzi we-enzyme ungancitshiswa yi-oxidant. I-DTT ingasabela kunye nee-oxidants ukuze inciphise izinto ezingenabungozi, ngaloo ndlela ikhusela umsebenzi kunye nokuzinza kwe-enzyme.
Xa kuthelekiswa nee-arhente zokunciphisa zemveli ezifana ne-β-mercaptoethanol (β-ME), i-DTT ithathwa njenge-arhente yokunciphisa ekhuselekileyo nezinzileyo. Ayizinzanga kuphela kwisisombululo samanzi, kodwa ikwagcina iipropati zayo zokunciphisa phantsi kobushushu obuphezulu kunye neemeko ze-asidi.
Ukusetyenziswa kwe-DTT kulula kakhulu. Ngokubanzi, i-DTT inyibilikiswa kwi-buffer efanelekileyo ize yongezwe kwinkqubo yovavanyo. Uxinzelelo olufanelekileyo lwe-DTT kufuneka lumiselwe ngokwesilingo esithile, kwaye lusetyenziswa ngokubanzi kuluhlu lwe-0.1-1mM. Uxinzelelo oluphantsi lunokunciphisa iziphumo ezimbi ekukhuleni kweseli kwaye lunokunciphisa ubuthi ngenxa yokwanda kweeproteni ekujoliswe kuzo. Uxinzelelo oluphezulu lunokubangela umthwalo omkhulu we-metabolic yeseli, okuchaphazela ukukhula kweseli kunye nokusebenza kakuhle kokubonakaliswa.
Indlela yokufumanisa uxinaniso olufanelekileyo ingaba kukuvavanya inqanaba lokubonakaliswa kweproteni ekujoliswe kuyo ngokwenza uvavanyo lokungeniswa kwe-IPTG kumanqanaba ahlukeneyo. Uvavanyo lokukhulisa inkcubeko oluncinci lunokwenziwa kusetyenziswa uluhlu lwamanqanaba e-IPTG (umz. 0.1 mM, 0.5 mM, 1 mM, njl.njl.) kwaye isiphumo sokubonakaliswa kumanqanaba ahlukeneyo sinokuvavanywa ngokufumanisa inqanaba lokubonakaliswa kweproteni ekujoliswe kuyo (umz. i-Western blot okanye i-fluorescence detection). Ngokweziphumo zovavanyo, uxinaniso olunesiphumo sokubonakaliswa esingcono lukhethwe njengoxinaniso olufanelekileyo.
Ukongeza, ungabhekisa kwiincwadi ezifanelekileyo okanye amava ezinye iilabhoratri ukuze uqonde uluhlu loxinzelelo lwe-IPTG olusetyenziswa rhoqo phantsi kweemeko ezifanayo zovavanyo, uze ulungise kwaye ulungelelanise ngokweemfuno zovavanyo.
Kubalulekile ukuqaphela ukuba uxinaniso olufanelekileyo lunokwahluka ngokuxhomekeke kwiinkqubo ezahlukeneyo zokubonakalisa, iiproteni ezijoliswe kuzo, kunye neemeko zovavanyo, ngoko ke kungcono ukwenza ngcono ngokwemeko nganye.
Ngamafutshane, i-DTT sisixhobo sokunciphisa esisetyenziswa rhoqo esinokusetyenziselwa ukunciphisa iibhondi ze-disulfide kwiiproteni nakwezinye ii-biomolecules kunye nokukhusela umsebenzi we-enzyme kunye nozinzo. Sisetyenziswe kakhulu kwiimvavanyo ze-biochemistry kunye ne-molecular biology.
Ixesha lokuthumela: Sep-28-2023