Dithiothreitol (DTT) ndi chochepetsera chomwe chimagwiritsidwa ntchito kwambiri, chomwe chimadziwikanso kuti chowonjezera chatsopano chobiriwira.Ndi gulu laling'ono la mamolekyu okhala ndi magulu awiri a mercaptan (-SH).Chifukwa chakuchepetsa kwake komanso kukhazikika kwake, DTT imagwiritsidwa ntchito kwambiri pakuyesa kwa biochemistry ndi ma cell biology.
Ntchito yayikulu ya DTT ndikuchepetsa zomangira za disulfide mu mapuloteni ndi ma biomolecules ena.Kugwirizana kwa disulfide ndi gawo lofunika kwambiri la mapuloteni opindika ndi kukhazikika, koma pansi pa zochitika zina zoyesera, monga kusanthula kwa SDS-PAGE kuchepetsedwa, kubwezeretsanso mapuloteni ndi kupukuta, ndikofunikira kuchepetsa mgwirizano wa disulfide kumagulu awiri a thiol kuti atsegule mawonekedwe a malo. mapuloteni.DTT ikhoza kuchitapo kanthu ndi zomangira za disulfide kuti ziwachepetse kumagulu a mercaptan, motero amatsegula mawonekedwe a malo a mapuloteni ndikupangitsa kuti zikhale zosavuta kusanthula ndi kuyendetsa.
DTT ingagwiritsidwenso ntchito kuteteza ntchito ya enzyme ndi kukhazikika.Muzochitika zina zomwe zimakhudzidwa ndi ma enzyme, ntchito ya enzyme imatha kuchepetsedwa ndi okosijeni.DTT imatha kuchitapo kanthu ndi okosijeni kuti iwachepetse kuzinthu zopanda vuto, potero kuteteza ntchito ndi kukhazikika kwa enzyme.
Poyerekeza ndi zochepetsera zachikhalidwe monga β-mercaptoethanol (β-ME), DTT imatengedwa kuti ndi yochepetsetsa yotetezeka komanso yokhazikika.Sili wokhazikika mu njira yamadzimadzi, komanso imasunga zinthu zake zochepetsera pansi pa kutentha kwakukulu ndi acid-base.
Kugwiritsa ntchito DTT ndikosavuta.Nthawi zambiri, DTT imasungunuka mu buffer yoyenera kenako ndikuwonjezeredwa ku dongosolo loyesera.Kuchuluka kwabwino kwa DTT kumafunika kutsimikiziridwa molingana ndi kuyesa kwapadera, ndipo nthawi zambiri amagwiritsidwa ntchito mumtundu wa 0.1-1mM.Kutsika pang'ono kumatha kuchepetsa zotsatira zoyipa pakukula kwa ma cell ndipo kumatha kuchepetsa cytotoxicity chifukwa chakuchulukirachulukira kwa mapuloteni omwe mukufuna.Kuchulukirachulukira kungayambitse kulemedwa kwambiri kwa ma cell, kusokoneza kukula kwa ma cell komanso magwiridwe antchito.
Njira yodziwira kukhazikika koyenera ingakhale kuyesa kuchuluka kwa mapuloteni omwe akuwunikira poyesa kuyesa kwa IPTG mosiyanasiyana.Mayeso ang'onoang'ono azikhalidwe amatha kuyesedwa pogwiritsa ntchito kuchuluka kwa IPTG (monga 0.1 mM, 0.5 mM, 1 mM, ndi zina zambiri.) komanso momwe mawuwo amamvekera pazigawo zosiyanasiyana akhoza kuyesedwa pozindikira kuchuluka kwa mawu a protein yomwe mukufuna (monga Western kuzindikira kwa blot kapena fluorescence).Malingana ndi zotsatira zoyesera, ndende yokhala ndi mawu abwino kwambiri inasankhidwa ngati ndende yabwino kwambiri.
Kuphatikiza apo, mutha kutchulanso zolemba zoyenera kapena zomwe zachitika m'ma laboratories ena kuti mumvetsetse kuchuluka kwa IPTG komwe amagwiritsidwa ntchito nthawi zambiri pansi pamiyeso yofananira, kenako kukhathamiritsa ndikusintha molingana ndi zosowa zoyeserera.
Ndikofunikira kuzindikira kuti kuwongolera koyenera kumatha kusiyanasiyana malinga ndi machitidwe osiyanasiyana ofotokozera, mapuloteni omwe mukufuna, ndi mikhalidwe yoyesera, kotero ndikwabwino kukhathamiritsa pazochitika ndi milandu.
Mwachidule, DTT ndi njira yochepetsera yomwe ingagwiritsidwe ntchito kuchepetsa zomangira za disulfide mu mapuloteni ndi ma biomolecules ena komanso kuteteza ntchito ya enzyme ndi kukhazikika.Zakhala zikugwiritsidwa ntchito kwambiri pakuyesa kwa biochemistry ndi molekyulu ya biology.
Nthawi yotumiza: Sep-28-2023