Dithiothreitol (DTT) ndi mankhwala ochepetsa omwe amagwiritsidwa ntchito kwambiri, omwe amadziwikanso kuti chowonjezera chatsopano chobiriwira. Ndi mankhwala ang'onoang'ono a organic okhala ndi magulu awiri a mercaptan (-SH). Chifukwa cha mphamvu zake zochepetsera komanso kukhazikika, DTT imagwiritsidwa ntchito kwambiri mu biochemistry ndi molecular biology.
Ntchito yaikulu ya DTT ndikuchepetsa ma disulfide bonds mu mapuloteni ndi ma biomolecule ena. Disulfide bond ndi gawo lofunika kwambiri pakupindika ndi kukhazikika kwa mapuloteni, koma pansi pa zochitika zina zoyesera, monga kusanthula kwa SDS-PAGE komwe kumachepetsedwa, kubwezeretsanso mapuloteni ndi kupindika, ndikofunikira kuchepetsa disulfide bond ku magulu awiri a thiol kuti mutsegule kapangidwe ka puloteni. DTT imatha kuchitapo kanthu ndi ma disulfide bonds kuti iwachepetse kukhala magulu a mercaptan, motero kutsegula kapangidwe ka puloteni komwe kumakhalapo ndikupangitsa kuti zikhale zosavuta kusanthula ndikusintha.
DTT ingagwiritsidwenso ntchito kuteteza ntchito ya ma enzyme ndi kukhazikika. Mu zochitika zina zomwe zimayambitsidwa ndi ma enzyme, ntchito ya enzyme ikhoza kuchepetsedwa ndi oxidant. DTT imatha kuchita ndi ma oxidant kuti iwachepetse kukhala zinthu zopanda vuto, potero kuteteza ntchito ndi kukhazikika kwa enzyme.
Poyerekeza ndi zinthu zochepetsera zinthu zakale monga β-mercaptoethanol (β-ME), DTT imaonedwa kuti ndi chinthu chochepetsera zinthu chotetezeka komanso chokhazikika. Sichimangokhala chokhazikika mumadzi, komanso chimasunga mphamvu zake zochepetsera zinthu pansi pa kutentha kwambiri komanso acid-base.
Kugwiritsa ntchito DTT n'kosavuta. Kawirikawiri, DTT imasungunuka mu buffer yoyenera kenako nkuwonjezeredwa ku dongosolo loyesera. Kuchuluka kwa DTT kuyenera kutsimikiziridwa malinga ndi kuyesera komweko, ndipo nthawi zambiri kumagwiritsidwa ntchito pa 0.1-1mM. Kuchuluka kochepa kumatha kuchepetsa zotsatira zoyipa pakukula kwa maselo ndipo kungachepetse poizoni chifukwa cha kuchuluka kwa mapuloteni omwe amafunidwa. Kuchuluka kwakukulu kungayambitse kuchuluka kwa kagayidwe ka maselo, zomwe zimakhudza kukula kwa maselo ndi magwiridwe antchito a maselo.
Njira yodziwira kuchuluka kwabwino kwambiri ingakhale kuyesa kuchuluka kwa mapuloteni omwe akufunidwa mwa kuchita mayeso oyambitsa IPTG pamlingo wosiyanasiyana. Mayeso ang'onoang'ono okulitsa akhoza kuchitidwa pogwiritsa ntchito mitundu yosiyanasiyana ya IPTG (monga 0.1 mM, 0.5 mM, 1 mM, ndi zina zotero) ndipo zotsatira za kuchuluka kwa mapuloteni pamlingo wosiyanasiyana zitha kuyesedwa pozindikira kuchuluka kwa mapuloteni omwe akufunidwa (monga Western blot kapena fluorescence detection). Malinga ndi zotsatira za kafukufuku, kuchuluka kwa mapuloteni omwe ali ndi mphamvu yabwino kwambiri yowonetsera kwasankhidwa ngati kuchuluka kwabwino kwambiri.
Kuphatikiza apo, mutha kuwonanso mabuku oyenera kapena zomwe zachitika m'ma laboratories ena kuti mumvetsetse kuchuluka kwa IPTG komwe kumagwiritsidwa ntchito kwambiri pansi pa mikhalidwe yofanana yoyesera, kenako ndikukonza ndikusintha malinga ndi zosowa zoyesera.
Ndikofunikira kudziwa kuti kuchuluka kwabwino kwambiri kumatha kusiyana malinga ndi machitidwe osiyanasiyana owonetsera, mapuloteni ofunikira, ndi mikhalidwe yoyesera, kotero ndibwino kukonza bwino nthawi ndi nthawi.
Mwachidule, DTT ndi mankhwala ochepetsa omwe amagwiritsidwa ntchito kwambiri omwe angagwiritsidwe ntchito kuchepetsa ma disulfide bond mu mapuloteni ndi ma biomolecule ena komanso kuteteza ntchito ya ma enzyme ndi kukhazikika kwawo. Yagwiritsidwa ntchito kwambiri mu biochemistry ndi ma molecular biology.
Nthawi yotumizira: Sep-28-2023