Kev ceev faj rau kev siv ultrafiltration raj
(1) Xaiv lub raj ultrafiltration uas tsim nyog. Feem ntau, qhov hnyav molecular yuav tsum khaws cia yuav tsum tsis txhob ntau tshaj 1/3 ntawm qhov hnyav molecular ntawm lub hom phiaj protein. Piv txwv li, yog tias qhov hnyav molecular ntawm lub hom phiaj protein yog 35kDa, lub raj ultrafiltration nrog 10kDa molecular hnyav tuaj yeem xaiv. Yog hais tias qhov hnyav molecular ntawm lub hom phiaj protein yog kwv yees li 10kd, lub raj ultrafiltration nrog ib qho hnyav molecular ntawm 3KD tuaj yeem siv. Ua tib zoo nyeem phau ntawv qhia thiab xyuam xim rau qhov sib txawv ntawm kev ua siab ntev ntawm ultrafiltration membrane rau ntau yam tshuaj.
(2) Cov khoom tshiab yuav ultrafiltration yog qhuav. Ntxiv dej ultrapure ua ntej siv, thiab dej yuav dhau los ntawm daim nyias nyias. Ua ntej txias nws nyob rau hauv ib da dej dej khov los yog lub tub yees rau ob peb feeb. Tom qab ntawd ncuav dej thiab ntxiv cov kua protein. Tus nqi ntxiv yuav tsum tsis pub tshaj cov kab dawb nyob rau sab saum toj ntawm lub raj. Kev ua haujlwm yuav tsum yog lub teeb. Ua ntej ntxiv cov tshuaj protein, lub raj ultrafiltration yuav tsum tau muab tso rau hauv dej khov rau precooling.
(3) Sib npaug. Ob qhov loj thiab qhov nruab nrab ntawm lub ntiajteb txawj nqus yuav tsum sib npaug. Nco ntsoov tias qhov ceev thiab acceleration yuav tsum tsis txhob ceev heev, txwv tsis pub lub ultrafiltration membrane yuav ncaj qha puas. Pib centrifugal ultrafiltration (centrifuge precooling rau 4 degrees). Tom qab RPM ntawm cov centrifuges sib txawv hloov mus rau G, nws txawv. Kho lub acceleration ntawm lub centrifuge mus rau qhov qis tshaj iav kom txo tau lub siab ntawm daim nyias nyias. Nco ntsoov tias koj yuav tsum tos kom txog thaum lub centrifuge ncav cuag lub hom phiaj ceev ua ntej tawm hauv lub centrifuge, txwv tsis pub thaum muaj teeb meem nrog lub centrifuge, koj tsis tuaj yeem nrog nws thawj zaug. Cov kev taw qhia ntawm daim nyias nyias thiab lub rotating ncej yuav tsum tau kho raws li cov lus qhia (nyob rau hauv cov ntaub ntawv ntawm lub kaum sab xis rotating centrifuge, lub membrane yog perpendicular mus rau lub ncej). Hauv kev siv tswv yim, qhov ceev yog feem ntau qis dua li hauv phau ntawv qhia, uas tuaj yeem ua rau lub neej ntev ntawm cov yeeb nkab centrifugal.
(4) Thaum nws yog concentrated rau qhov seem 1ml, noj 50ul buffer tov thiab ntxiv 10ul ntws los mus saib seb nws tig xiav, thiaj li txiav txim seb lub ultrafiltration raj xau protein. Yog tias lub raj xau, ncuav cov txheej sab saud thiab ntws los ntawm lub raj tshiab thiab pib ultrafiltration. Txhawm rau txiav txim siab seb lub raj xau, centrifuge nrog 5mgml BSA rau 10min, tom qab ntawd siv cov dej ntws los, khiav cov kua nplaum protein lossis Bradford rau kev ntsuas ntxhib, thiab txuas ntxiv ntxiv cov kua protein ntxiv rau cov concentration (ua haujlwm rau dej khov kom tsis txhob muaj cov protein ntau. rhuab) kom txog thaum tag nrho cov kev daws teeb meem ntxiv. Ua tib zoo saib seb puas muaj cov nag lossis daus tshwm sim thaum lub sij hawm centrifugation, ua rau cov yeeb nkab txhaws. Yog tias nag lossis daus tshwm sim, txiav txim siab qhov ua rau nag lossis daus, txawm tias cov protein ntau dhau los yog qhov tsis tsim nyog; Cov qub tuaj yeem daws tau los ntawm ib txhij ultrafiltration nrog ntau lub raj ultrafiltration kom txo cov concentration. Cov tom kawg yog hloov cov kev daws teeb meem sib txawv kom txog thaum cov protein tsis tuaj yeem ua rau.
(5) Cov kauj ruam dhau los yog siv los mloog cov protein. Yog hais tias koj xav hloov qhov tsis, thaum tag nrho cov protein tov yog concentrated txog 1ml, maj mam ntxiv ib tug tshiab buffer (ultrafiltration los ntawm 0.22um ultrafiltration membrane), thiab ces mloog zoo txog li 1ml rau peb zaug sib law liag. Qhov kawg ntim ntawm qhov kawg concentration yog nyob ntawm qhov yuav tsum tau muaj protein ntau concentration, uas feem ntau tsis ntau tshaj 500ul, los yog nws yuav tsum tau concentrated tsawg tshaj li 200ul. Raws li qhov ntim ntawm qhov tsawg kawg nkaus 10 zaug txhua lub sijhawm, nws tuaj yeem ncav cuag ntau dua 1000 zaug peb zaug, uas tuaj yeem ua tiav lub hom phiaj ntawm kev hloov pauv tsis.
