Kev siv thiab ceev faj ntawm ultrafiltration centrifuge raj
1) Xaiv lub raj zoo ultrafiltration.Nco ntsoov tias UF daim nyias nyias txawv ntawm lawv cov qib siab kam rau ntau yam tshuaj.Feem ntau, cov hlab ntsha ultrafiltration nrog 10 kDa molecular hnyav txiav tawm tuaj yeem xaiv nrog qhov hnyav molecular txiav tawm uas yuav tsum tsis txhob siab dua 1/3 ntawm qhov hnyav molecular ntawm cov protein uas nyiam, xws li 35 kDa.Yog tias qhov hnyav molecular ntawm cov protein txaus siab nyob ib puag ncig 10 kd, lub raj ultrafiltration nrog lub qhov hnyav molecular txiav ntawm 3 KD tuaj yeem siv.
(2) Lub ultrafiltration, freshly muas, yog qhuav, nrog ultrapure dej ntxiv ua ntej siv thiab dej tag nrho dhau los ntawm daim nyias nyias, dej khov da dej los yog lub tub yees ua ntej txias rau ob peb feeb.Tom qab ntawd cov dej yog nchuav tawm, piv txwv li cov kua protein, thiab ntxiv ntau npaum li cas, qhov twg tsis ntau tshaj li cov kab dawb nyob rau saum lub raj.Kev ua haujlwm yog lub teeb, thiab lub raj ultrafiltration yuav tsum tau precooled ntawm cov dej khov ua ntej ntxiv cov kua protein.
3) Ob qhov loj thiab qhov nruab nrab ntawm lub ntiajteb txawj nqus tau mus txog qhov sib npaug.Nco ntsoov qhov kev sib hloov ceev thiab acceleration tsis ceev heev, txwv tsis pub ncaj qha ua rau lub ultrafiltration membrane.Centrifugal ultrafiltration tau pib (centrifuge precooled rau 4 degrees).Tom qab RPM ntawm cov centrifuges sib txawv tau hloov mus rau g, nws txawv.Lub acceleration ntawm lub centrifuge tau hloov mus rau qhov tsawg kawg nkaus, txo qhov siab ntawm daim nyias nyias.Nco ntsoov, nco ntsoov tawm ntawm lub centrifuge tom qab lub centrifuge tau mus txog nws qhov chaw ceev, lossis yog tias koj muaj teeb meem, koj tuaj yeem ua tsis tau nws thawj zaug.Kev taw qhia ntawm daim nyias nyias mus rau spindle tau hloov kho raws li cov lus qhia (angular centrifuge case yog membrane rau axis perpendicular).Hauv kev siv tswv yim, qhov kev sib hloov dav dav yog qis dua li cov lus qhia, kom lub neej ntawm lub raj centrifuge tuaj yeem txuas ntxiv.
3) Ob qhov loj thiab qhov nruab nrab ntawm lub ntiajteb txawj nqus tau mus txog qhov sib npaug.Nco ntsoov qhov kev sib hloov ceev thiab acceleration tsis ceev heev, txwv tsis pub ncaj qha ua rau lub ultrafiltration membrane.Centrifugal ultrafiltration tau pib (centrifuge precooled rau 4 degrees).Tom qab RPM ntawm cov centrifuges sib txawv tau hloov mus rau g, nws txawv.Lub acceleration ntawm centrifuge tau hloov mus rau qhov tsawg kawg nkaus, txo qhov siab ntawm daim nyias nyias.Nco ntsoov, nco ntsoov tawm ntawm lub centrifuge tom qab lub centrifuge tau mus txog nws qhov chaw ceev, lossis yog tias koj muaj teeb meem, koj tuaj yeem ua tsis tau nws thawj zaug.Kev taw qhia ntawm daim nyias nyias mus rau spindle tau hloov kho raws li cov lus qhia (angular centrifuge case yog membrane rau axis perpendicular).Hauv kev siv tswv yim, kev sib hloov dav dav yog qis dua li cov lus qhia, kom lub neej ntawm lub raj centrifuge tuaj yeem txuas ntxiv.
(4) Thaum concentrated rau qhov seem 1ml, noj 50ul ntawm kev daws teeb meem, ntxiv 10ul ntawm ntws los ntawm thiab saib seb puas muaj xim xiav, raws li kev txiav txim siab seb lub raj ultrafiltration puas muaj protein ntau.Yog hais tias lub raj yog leaky, rov ncuav lub txheej sab sauv thiab ntws mus rau hauv lub raj tshiab kom pib ultrafiltration.Txhawm rau txiav txim siab yog tias cov hlab ntsha tsis tuaj, centrifuge rau 10min nrog 5mgml ntawm BSA ua ntej flowthrough, khiav ntawm cov kua nplaum protein, los yog Bradford crude assay, thiab txuas ntxiv los ntawm kev ntxiv cov ntsiab lus protein ntxiv (uas ua haujlwm rau dej khov thiab tiv thaiv cov proteins los ntawm cua sov) kom txog rau thaum tag nrho cov concentrate tau ntxiv.Saib xyuas thaum lub sijhawm centrifugation yog nag lossis daus ntawm cov protein tshwm sim, ua rau lub raj kaw.Yog tias nag lossis daus tshwm sim, txiav txim siab seb qhov ua rau nag lossis daus yog qhov muaj protein ntau ntau lossis tsis tsim nyog;Cov qub tuaj yeem daws tau los ntawm ultrafiltration ntawm ntau lub raj ultrafiltration ib txhij, txo cov concentration, thiab tom kawg los ntawm kev sib pauv cov kev daws teeb meem sib txawv kom txog thaum tsis muaj nag lossis daus ntawm cov protein tshwm sim.
(4) Thaum concentrated rau qhov seem 1ml, noj 50ul ntawm kev daws teeb meem, ntxiv 10ul ntawm ntws los ntawm thiab saib seb puas muaj xim xiav, raws li kev txiav txim siab seb lub raj ultrafiltration puas muaj protein ntau.Yog hais tias lub raj yog leaky, rov ncuav lub txheej sab sauv thiab ntws mus rau hauv lub raj tshiab kom pib ultrafiltration.Txhawm rau txiav txim siab yog tias cov hlab ntsha tsis tuaj, centrifuge rau 10min nrog 5mgml ntawm BSA ua ntej flowthrough, khiav ntawm cov kua nplaum protein, los yog Bradford crude assay, thiab txuas ntxiv los ntawm kev ntxiv cov ntsiab lus protein ntxiv (uas ua haujlwm rau dej khov thiab tiv thaiv cov proteins los ntawm cua sov) kom txog rau thaum tag nrho cov concentrate tau ntxiv.Saib xyuas thaum lub sijhawm centrifugation yog nag lossis daus ntawm cov protein tshwm sim, ua rau lub raj kaw.Yog tias nag lossis daus tshwm sim, txiav txim siab seb qhov ua rau nag lossis daus yog qhov muaj protein ntau ntau lossis tsis tsim nyog;Cov qub tuaj yeem daws tau los ntawm ultrafiltration ntawm ntau lub raj ultrafiltration ib txhij, txo cov concentration, thiab tom kawg los ntawm kev sib pauv cov kev daws teeb meem sib txawv kom txog thaum tsis muaj nag lossis daus ntawm cov protein tshwm sim.
(5) Thawj ob peb kauj ruam yog siv los ua kom pom tseeb cov protein, thiab yog tias yuav tsum tsis txhob hloov pauv, maj mam ntxiv cov khoom tshiab (ultrafiltration los ntawm 0.22um ultrafiltration membrane) txog li 1ml ntawm tag nrho cov protein, thiab reconcentrate txog li 1ml rau peb. sib law liag, nrog rau qhov kawg concentrated kawg ntim nyob ntawm qhov xav tau protein concentration, feem ntau tsis ntau tshaj 500ul, tab sis kuj mus rau hauv 200ul.Ua kom tiav 1000 × lossis ntau dua rau peb lub sijhawm, qhov tseem ceeb npaum li qhov hloov pauv tsis zoo, raws li suav los ntawm tsawg kawg 10 × lossis yog li ntim ntxiv txhua lub sijhawm.
(5) Thawj ob peb kauj ruam yog siv los ua kom pom tseeb cov protein, thiab yog tias yuav tsum tsis txhob hloov pauv, maj mam ntxiv cov khoom tshiab (ultrafiltration los ntawm 0.22um ultrafiltration membrane) txog li 1ml ntawm tag nrho cov protein, thiab reconcentrate txog li 1ml rau peb. sib law liag, nrog rau qhov kawg concentrated kawg ntim nyob ntawm qhov xav tau protein concentration, feem ntau tsis ntau tshaj 500ul, tab sis kuj mus rau hauv 200ul.Ua kom tiav 1000 × lossis ntau dua rau peb lub sijhawm, qhov tseem ceeb npaum li qhov hloov pauv tsis zoo, raws li suav los ntawm tsawg kawg 10 × lossis yog li ntim ntxiv txhua lub sijhawm.
Post lub sij hawm: Nov-09-2022