Ko te Dithiothreitol (DTT) he matū whakaiti e whakamahia whānuitia ana, e mōhiotia ana ko te tāpiri matomato hou. He matū waro ngota iti me ngā rōpū mercaptan e rua (-SH). Nā ōna āhuatanga whakaiti me te pumau, e whakamahia whānuitia ana te DTT i roto i te matū koiora me ngā whakamātautau koiora ngota.
Ko te mahi matua a te DTT he whakaiti i ngā here disulfide i roto i ngā pūmua me ētahi atu ngota koiora. He wāhanga nui te here disulfide o te takai me te pumau o te pūmua, engari i raro i ētahi āhuatanga whakamātautau, pērā i te tātari SDS-PAGE whakaitiiti, te whakakotahitanga pūmua me te takai, me whakaiti te here disulfide ki ngā rōpū thiol e rua hei wetewete i te hanganga ā-wāhi o te pūmua. Ka taea e te DTT te tauhohe ki ngā here disulfide hei whakaiti i a rātou ki ngā rōpū mercaptan, ā, ka whakatuwhera i te hanganga ā-wāhi o te pūmua, ā, ka ngāwari te tātari me te whakahaere.
Ka taea hoki te whakamahi i te DTT hei tiaki i te mahi me te pumau o te whākōkī. I roto i ētahi tauhohenga whakakorikori whākōkī, ka taea te whakaiti i te mahi a te whākōkī e te waikura. Ka taea e te DTT te tauhohe ki ngā waikura hei whakaiti i a rātou hei matū kore kino, ā, mā reira ka tiakina te mahi me te pumau o te whākōkī.
Ki te whakatauritea ki ngā kaiwhakaiti tuku iho pērā i te β-mercaptoethanol (β-ME), e kiia ana ko te DTT he kaiwhakaiti haumaru ake, he pumau ake hoki. Ehara i te mea he pumau noa i roto i te wairewa, engari ka mau tonu hoki ōna āhuatanga whakaiti i raro i ngā āhuatanga pāmahana teitei me te waikawa-pūtake.
He tino māmā te whakamahinga o te DTT. I te nuinga o te wā, ka whakarewaina te DTT ki roto i tētahi pūhui e tika ana, kātahi ka tāpirihia ki te pūnaha whakamātautau. Me whakatau te kukū tino pai o te DTT i runga i te whakamātautau motuhake, ā, e whakamahia ana i te whānuitanga o te 0.1-1mM. Ka taea e ngā kukū iti te whakaiti i ngā pānga kino ki te tipu o te pūtau, ā, ka taea hoki te whakaiti i te paitini pūtau nā te nui rawa o te whakaputanga o ngā pūmua ūnga. Ka taea e ngā kukū teitei ake te whakararu i te taumahatanga pākia o te pūtau, ka pā ki te tipu me te whai huatanga o te whakaputanga.
Ko te huarahi ki te whakatau i te kukū tino pai ko te aromatawai i te taumata whakapuakanga o te pūmua ūnga mā te whakahaere i ngā whakamātautau whakaoho IPTG i ngā kukū rerekē. Ka taea te whakahaere i ngā whakamātautau ahurea iti mā te whakamahi i te whānuitanga o ngā kukū IPTG (hei tauira, 0.1 mM, 0.5 mM, 1 mM, me ētahi atu) ā, ka taea te aromatawai i te pānga whakapuakanga i ngā kukū rerekē mā te kimi i te taumata whakapuakanga o te pūmua ūnga (hei tauira, te Western blot, te kitenga fluorescence rānei). E ai ki ngā hua o te whakamātautau, ko te kukū me te pānga whakapuakanga pai rawa atu i tīpakohia hei kukū tino pai.
Hei tāpiritanga, ka taea hoki e koe te tirotiro i ngā tuhinga e tika ana, i ngā wheako rānei o ētahi atu taiwhanga hei mārama ki te awhe kukū IPTG e whakamahia whānuitia ana i raro i ngā āhuatanga whakamātautau ōrite, kātahi ka arotau me te whakarerekē kia rite ki ngā hiahia whakamātautau.
He mea nui kia mōhiotia ko te kukū tino pai ka rerekē pea i runga i ngā pūnaha whakapuakitanga rerekē, ngā pūmua ūnga, me ngā āhuatanga whakamātautau, nō reira he pai ake te arotau i runga i te kaupapa takitahi.
Hei whakarāpopototanga, he matū whakaiti te DTT e whakamahia whānuitia ana hei whakaiti i ngā here disulfide i roto i ngā pūmua me ētahi atu koiora ngota, hei tiaki hoki i te mahi me te pumau o ngā whākōkī. Kua whakamahia whānuitia i roto i ngā whakamātautau matū koiora me te koiora ngota.
Wā tuku: Hepetema-28-2023