Dithiothreitol (DTT) ke sesebelisoa se fokotsang se sebelisoang haholo, se tsejoang hape e le sehlomathiso se secha se setala. Ke motsoako o monyane oa limolek'hule tse phelang o nang le lihlopha tse peli tsa mercaptan (-SH). Ka lebaka la thepa ea eona e fokotsang le botsitso, DTT e sebelisoa haholo litekong tsa biochemistry le baeloji ea limolek'hule.
Karolo e ka sehloohong ea DTT ke ho fokotsa litlamo tsa disulfide liprotheineng le limolek'huleng tse ling. Tlamo ea disulfide ke karolo ea bohlokoa ea ho mena le botsitso ba protheine, empa tlas'a maemo a itseng a liteko, joalo ka tlhahlobo ea SDS-PAGE e fokotsoang, ho kopanya protheine hape le ho mena, hoa hlokahala ho fokotsa tlamo ea disulfide ho lihlopha tse peli tsa thiol ho hlakola sebopeho sa sebaka sa protheine. DTT e ka arabela ka litlamo tsa disulfide ho li fokotsa ho ea ho lihlopha tsa mercaptan, ka hona e bula sebopeho sa sebaka sa protheine le ho etsa hore ho be bonolo ho se sekaseka le ho se laola.
DTT e ka boela ea sebelisoa ho sireletsa ts'ebetso ea li-enzyme le botsitso. Likarabelong tse ling tse susumetsoang ke li-enzyme, ts'ebetso ea li-enzyme e ka fokotsoa ke oxidant. DTT e ka arabela le li-oxidant ho li fokotsa ho ba lintho tse se nang kotsi, ka hona e sireletsa ts'ebetso le botsitso ba li-enzyme.
Ha e bapisoa le metsoako ea setso ea ho fokotsa e kang β-mercaptoethanol (β-ME), DTT e nkoa e le moemeli oa ho fokotsa o sireletsehileng le o tsitsitseng haholoanyane. Ha e tsitse feela ka har'a tharollo ea metsi, empa hape e boloka thepa ea eona ea ho fokotsa tlas'a maemo a mocheso o phahameng le a acid-base.
Tšebeliso ea DTT e bonolo haholo. Ka kakaretso, DTT e qhibilihisoa ka har'a buffer e loketseng ebe e eketsoa tsamaisong ea liteko. Khatello e ntle ka ho fetisisa ea DTT e hloka ho fumanoa ho latela teko e itseng, 'me hangata e sebelisoa ho tloha ho 0.1-1mM. Khatello e tlase e ka fokotsa litlamorao tse mpe kholong ea sele 'me ea fokotsa chefo e bakoang ke ho eketseha ha liprotheine tse reriloeng. Khatello e phahameng e ka baka moroalo o feteletseng oa metabolism ea sele, ea ama kholo ea sele le katleho ea tlhahiso.
Mokhoa oa ho fumana hore na ho na le mahloriso a nepahetseng e ka ba ho lekola boemo ba tlhahiso ea protheine e reretsoeng ka ho etsa liteko tsa ho kenya IPTG maemong a fapaneng. Liteko tsa temo tse nyane li ka etsoa ho sebelisoa mefuta e fapaneng ea mahloriso a IPTG (mohlala, 0.1 mM, 0.5 mM, 1 mM, jj.) 'me phello ea tlhahiso maemong a fapaneng e ka lekoa ka ho lemoha boemo ba tlhahiso ea protheine e reretsoeng (mohlala, Western blot kapa fluorescence detection). Ho ea ka liphetho tsa liteko, mahloriso a nang le phello e ntle ka ho fetisisa ea tlhahiso a khethiloe e le mahloriso a nepahetseng ka ho fetisisa.
Ho phaella moo, o ka boela wa sheba dingolwa tse amehang kapa phihlelo ya dilaboratori tse ding ho utlwisisa mefuta e sebediswang haholo ya mahloriso a IPTG tlasa maemo a tshwanang a teko, ebe o ntlafatsa le ho fetola ho ya ka ditlhoko tsa teko.
Ho bohlokoa ho hlokomela hore mahloriso a nepahetseng a ka fapana ho latela litsamaiso tse fapaneng tsa tlhahiso, liprotheine tse reriloeng, le maemo a liteko, kahoo ho molemo ho ntlafatsa ho latela nyeoe ka 'ngoe.
Ka bokhutšoanyane, DTT ke sesebelisoa se fokotsang se sebelisoang haholo se ka sebelisoang ho fokotsa litlamo tsa disulfide liprotheineng le limolek'huleng tse ling le ho sireletsa ts'ebetso ea li-enzyme le botsitso. E sebelisitsoe haholo litekong tsa biochemistry le baeloji ea limolek'hule.
Nako ea poso: Loetse-28-2023