Indlela yokuchonga ugxininiso olufanelekileyo?
Kwi-inducer IPTG (isopropyl-beta-d-thiogalactoside), okukhona uxinzelelo luphezulu, kokukhona lungcono. Uxinzelelo olufanelekileyo luxhomekeke kwiimeko ezithile zovavanyo kunye nesiphumo sokungeniswa esifunekayo.
Ngokubanzi, uxinzelelo lwe-IPTG lusetyenziswa kuluhlu lwe-0.1-1 mM. Uxinzelelo oluphantsi lunokunciphisa iziphumo ezibi ekukhuleni kweeseli kwaye lunokunciphisa ubuthi obuvela kwi-cytotoxicity ngenxa yokwanda kweeproteni ekujoliswe kuzo. Uxinzelelo oluphezulu lunokubangela umthwalo omkhulu we-metabolism yeeseli, nto leyo echaphazela ukukhula kweeseli kunye nokusebenza kakuhle kokubonakaliswa kwazo.
Indlela yokufumanisa uxinaniso olufanelekileyo ingaba kukuvavanya inqanaba lokubonakaliswa kweproteni ekujoliswe kuyo ngokwenza uvavanyo lokungeniswa kwe-IPTG kumanqanaba ahlukeneyo. Uvavanyo lokukhulisa inkcubeko oluncinci lunokwenziwa kusetyenziswa uluhlu lwamanqanaba e-IPTG (umz. 0.1 mM, 0.5 mM, 1 mM, njl.njl.) kwaye isiphumo sokubonakaliswa kumanqanaba ahlukeneyo sinokuvavanywa ngokufumanisa inqanaba lokubonakaliswa kweproteni ekujoliswe kuyo (umz. i-Western blot okanye i-fluorescence detection). Ngokweziphumo zovavanyo, uxinaniso olunesiphumo sokubonakaliswa esingcono lukhethwe njengoxinaniso olufanelekileyo.
Ukongeza, ungabhekisa kwiincwadi ezifanelekileyo okanye amava ezinye iilabhoratri ukuze uqonde uluhlu loxinzelelo lwe-IPTG olusetyenziswa rhoqo phantsi kweemeko ezifanayo zovavanyo, uze ulungise kwaye ulungelelanise ngokweemfuno zovavanyo.
Kubalulekile ukuqaphela ukuba uxinaniso olufanelekileyo lunokwahluka ngokuxhomekeke kwiinkqubo ezahlukeneyo zokubonakalisa, iiproteni ezijoliswe kuzo, kunye neemeko zovavanyo, ngoko ke kungcono ukwenza ngcono ngokwemeko nganye.
Ixesha lokuthumela: Sep-28-2023