I-Dithiothreitol (DTT) iyi-ejenti yokunciphisa esetshenziswa kakhulu, eyaziwa nangokuthi isithasiselo esisha esiluhlaza. Iyinhlanganisela encane ye-molecule organic enamaqembu amabili e-mercaptan (-SH). Ngenxa yezakhiwo zayo zokunciphisa kanye nokuqina, i-DTT isetshenziswa kabanzi ekuhlolweni kwe-biochemistry kanye ne-molecular biology.
Indima eyinhloko ye-DTT ukunciphisa izibopho ze-disulfide kumaprotheni nakwamanye ama-biomolecule. Isibopho se-disulfide siyingxenye ebalulekile yokugoqwa nokuqina kwamaprotheni, kodwa ngaphansi kwezimo ezithile zokuhlola, njengokuhlaziywa kwe-SDS-PAGE okuncishiswayo, ukuhlanganiswa kwamaprotheni nokugoqwa, kuyadingeka ukunciphisa isibopho se-disulfide kumaqembu amabili e-thiol ukuze kuhlukaniswe isakhiwo sendawo seprotheyini. I-DTT ingasabela ngezibopho ze-disulfide ukuze inciphise zibe amaqembu e-mercaptan, ngaleyo ndlela ivule isakhiwo sendawo seprotheyini futhi kwenze kube lula ukuyihlaziya nokuyilawula.
I-DTT ingasetshenziswa futhi ukuvikela umsebenzi we-enzyme kanye nokuzinza. Kwezinye izindlela zokusabela ezibangelwa yi-enzyme, umsebenzi we-enzyme ungancishiswa yi-oxidant. I-DTT ingasabela nama-oxidant ukuze iwanciphise abe yizinto ezingenangozi, ngaleyo ndlela ivikele umsebenzi kanye nokuzinza kwe-enzyme.
Uma kuqhathaniswa nama-reducing agents endabuko njenge-β-mercaptoethanol (β-ME), i-DTT ibhekwa njenge-reducing agent ephephile futhi ezinzile. Ayigcini nje ngokuqina esixazululweni samanzi, kodwa futhi igcina izakhiwo zayo zokunciphisa ngaphansi kwezimo zokushisa okuphezulu kanye ne-acid-base.
Ukusetshenziswa kwe-DTT kulula kakhulu. Ngokuvamile, i-DTT incibilikiswa ku-buffer efanele bese ifakwa ohlelweni lokuhlola. Ukuhlushwa okungcono kakhulu kwe-DTT kudinga ukunqunywa ngokuya ngokuhlolwa okuthile, futhi ngokuvamile kusetshenziswa ebangeni eliphakathi kuka-0.1-1mM. Ukuhlushwa okuphansi kunganciphisa imiphumela emibi ekukhuleni kwamaseli futhi kunganciphisa ubuthi obuvela kuma-cytotoxicity ngenxa yokwanda kwamaprotheni aqondiwe. Ukuhlushwa okuphezulu kungabangela umthwalo omkhulu we-metabolic wamaseli, okuthinta ukukhula kwamaseli kanye nokusebenza kahle kokuvezwa.
Indlela yokuthola ukuhlushwa okungcono kakhulu kungaba ukuhlola izinga lokuvezwa kweprotheyini eqondiwe ngokwenza izivivinyo zokungeniswa kwe-IPTG ngamanani ahlukene. Izivivinyo zokulima ezincane zingenziwa kusetshenziswa ububanzi bamanani e-IPTG (isb. 0.1 mM, 0.5 mM, 1 mM, njll.) kanye nomphumela wokuvezwa ngamanani ahlukene ungahlolwa ngokuthola izinga lokuvezwa kweprotheyini eqondiwe (isb. i-Western blot noma ukutholwa kwe-fluorescence). Ngokusho kwemiphumela yokuhlola, ukuhlushwa okunomphumela omuhle kakhulu wokuvezwa kukhethwe njengokuhlushwa okuhle kakhulu.
Ngaphezu kwalokho, ungabhekisela nasezincwadini ezifanele noma kokuhlangenwe nakho kwamanye amalabhorethri ukuze uqonde ububanzi bokuhlushwa kwe-IPTG obusetshenziswa njalo ngaphansi kwezimo ezifanayo zokuhlola, bese wenza ngcono futhi ulungise ngokwezidingo zokuhlola.
Kubalulekile ukuqaphela ukuthi ukuhlushwa okuhle kakhulu kungahluka kuye ngezinhlelo ezahlukene zokuveza, amaprotheni aqondiwe, kanye nezimo zokuhlola, ngakho-ke kungcono ukwenza ngcono ngokwesisekelo secala ngalinye.
Ngamafuphi, i-DTT iyi-ejenti yokunciphisa esetshenziswa kakhulu engasetshenziswa ukunciphisa izibopho ze-disulfide kumaprotheni nakwamanye ama-biomolecule kanye nokuvikela umsebenzi we-enzyme kanye nokuzinza. Isetshenziswe kabanzi ekuhlolweni kwe-biochemistry kanye ne-molecular biology.
Isikhathi sokuthunyelwe: Septhemba-28-2023