Ngaba i-centrifuge yahlula i-DNA kwiiproteni

Ngaba iCentrifuge iyahlula iDNA kwiiProteni?

Intshayelelo

I-Centrifugation yindlela ebalulekileyo esetyenziswa kakhulu kuphando lwesayensi nakwiilabhoratri zeklinikhi ukwahlula iinxalenye ezahlukeneyo zesampuli ngokusekelwe kubuninzi bazo. Enye indlela eqhelekileyo yokusebenzisa le ndlela ibandakanya ukwahlula i-DNA kunye neeproteni kwisampuli ethile yebhayoloji. Kweli nqaku, siza kuhlola ukuba i-centrifuge iyakwazi na ukwahlula i-DNA kwiiproteni ngempumelelo kwaye sijonge iindlela ezahlukeneyo ezisetyenziswayo ukufezekisa ukwahlukana okusebenzayo.

Ukuqonda i-Centrifugation

Ngaphambi kokuba sijonge kwiinkcukacha zokwahlula i-DNA kwiiproteni sisebenzisa i-centrifuge, kubalulekile ukuqonda imigaqo esisiseko ye-centrifuge. I-centrifuge isebenza ngokomgaqo we-sedimentation kwaye isebenzisa ukujikeleza okukhawulezayo ukudala amandla amakhulu e-centrifugal. La mandla abangela ukuba amasuntswana kwisampulu ahlukane ngokusekelwe kubuninzi bawo, kunye namasuntswana axineneyo afudukela ezantsi kwityhubhu yesampulu. Ngenxa yoko, amasuntswana akhanyayo ahlala phezulu.

Umngeni Wokwahlula iDNA kunye neeProteni

Xa kufikwa ekwahlukaniseni iDNA kunye neeproteni, oku kudala umceli mngeni kuba zombini iimolekyuli zinesisindo esifanayo seemolekyuli. Ngaphandle kwendlela yokwahlulahlula esebenzayo, kunzima ukufumana iisampulu zeDNA okanye zeproteni ezicocekileyo. Nangona kunjalo, i-centrifugation inokunceda kwinkqubo yokwahlulahlula, kodwa isenokunganeli yodwa.

1. Ukwahlulahlula kweCentrifugation: Ukwahlulwa kokuqala

2. I-Ultracentrifugation: Ulwahlulo oluphuculweyo

3. I-Centrifugation ye-Density Gradient: Ukuhlaziya iNkqubo

4. Ukubaluleka koKhetho lweBuffer

5. Ukudibanisa i-Centrifugation ngokuchasene nezinye iindlela zokwahlulahlula

I-Centrifugation yokwahluka: Ukwahlukana kokuqala

I-differential centrifugation linyathelo lokuqala lokwahlula i-DNA kwiiproteni. Le ndlela isebenzisa isantya esahlukileyo se-centrifugation ukufezekisa ukwahlukana okubanzi kwezixhobo zeseli. Ngexesha lale nkqubo, iiseli ezihomogenized okanye iisampuli zezicubu zifakwa kwi-low-speed centrifugation, ngokuqhelekileyo malunga ne-1000-5000 revolutions ngomzuzu (rpm). Olu jikelezo oluphantsi lwenza kube lula ukwahlula ii-nuclei kunye neeseli ezipheleleyo, nto leyo ekhokelela kwi-crude fractionation. Nangona kunjalo, ayiboneleli ngokwahlukana okupheleleyo kwe-DNA kunye neeproteni.

I-Ultracentrifugation: Ulwahlulo oluphuculweyo

Ukuze koyiswe imida ye-differential centrifugation, i-ultracentrifugation ingena kwindawo. Ii-Ultracentrifuges zii-high-speed centrifuges ezikwaziyo ukufikelela kwisantya esifikelela kwi-100,000 rpm. Ngokubeka i-crude fraction efunyenwe kwi-low-speed centrifugation yokuqala ukuya kwi-ultracentrifugation, inqanaba eliphezulu lokwahlukana phakathi kwe-DNA kunye neeproteni linokuphunyezwa. Iimolekyuli ze-DNA ezinzima ziyanyibilika ziye ezantsi kwetyhubhu, zenze i-pellet ebonakalayo, ngelixa iiproteni zihlala kwi-supernatant.

I-Centrifugation ye-Density Gradient: Ukuhlaziya iNkqubo

Ukuze kuphuculwe ngakumbi ukwahlukana kwe-DNA kunye neeproteni, kusetyenziswa i-density gradient centrifugation. Kule ndlela, kulungiswa i-density gradient medium, efana ne-sucrose okanye i-cesium chloride. Le medium ineentlobo ngeentlobo zoxinano, olunyuka ukusuka phezulu ukuya ezantsi kwetyhubhu ye-centrifuge. Iqhekeza eliluhlaza elifunyenwe kwinyathelo langaphambili libekwa ngononophelo phezu kwe-density gradient kwaye liphinde lifakwe kwi-ultracentrifugation kwakhona. I-DNA kunye neeproteni zahlukana ngokusekelwe kuxinano lwazo kwaye zenze iibhendi ezahlukeneyo ngaphakathi kwe-gradient. Ukuqhekezwa kwe-gradient kuvumela ukubuyiselwa kweesampuli ze-DNA kunye neeproteni ezicocekileyo.

Ukubaluleka koKhetho lweBuffer

I-buffer esetyenziswa ngexesha le-centrifugation idlala indima ebalulekileyo ekufezekiseni ukwahlukana okusebenzayo. Ichaphazela uzinzo kunye nokuthembeka kwee-molecules ezahlulwe. Kwi-DNA, i-Tris-EDTA buffer ene-pH ye-8.0 isetyenziswa rhoqo ngenxa yokukwazi kwayo ukugcina uzinzo lwe-DNA. Kwelinye icala, iiproteni zifuna ii-buffers ezinezinga elithile le-pH, ngokuqhelekileyo malunga ne-7.0, ukuthintela ukubola kunye nokugcina isakhiwo sazo sendalo. Ukukhetha i-buffer efanelekileyo kuqinisekisa ukwahlukana ngokufanelekileyo kwe-DNA kunye neeproteni ngaphandle kokubeka umgangatho wazo esichengeni.

I-Centrifugation ngokuchasene nezinye iindlela zokwahlulahlula

Nangona i-centrifugation yindlela esetyenziswa kakhulu ekwahlukaniseni i-DNA kwiiproteni, kubalulekile ukuvuma ukuba isenokungabi yindlela yodwa esetyenziswayo. Ezinye iindlela, ezifana ne-chromatography, i-electrophoresis, kunye ne-precipitation, zinokuncedisa i-centrifugation ukuze kufunyanwe ukwahlukana okucokisekileyo. Indlela nganye ineenzuzo kunye nemida yayo, kwaye abaphandi badla ngokudibanisa iindlela ezininzi ukuze bafumane ubumsulwa obufunekayo kunye nokubuyiselwa kwe-DNA kunye neeproteni.

Isiphelo

Ukuqukumbela, nangona i-centrifuge iluncedo ngokwenene ekwahlukaniseni i-DNA kwiiproteni, isebenza kakuhle xa idibene nezinye iindlela. I-differential centrifugation, i-ultracentrifugation, kunye ne-density gradient centrifugation zenza uthotho lwamanyathelo okwahlula anceda ekufumaneni iisampulu ezicocekileyo. Ukongeza, ukukhetha ii-buffers ezifanelekileyo kunye nokuqwalasela ezinye iindlela zokwahlula kuphucula ngakumbi ukusebenza kakuhle kokwahlulwa kwe-DNA-protein.