Yuav Ua Li Cas Xaiv Qhov Tsim Nyog Ultrafiltration Tube
Ultrafiltration raj feem ntau siv rau cov protein ntau thiab tsis hloov pauv feem ntau yog siv hauv cov raj ultrafiltration. Muaj lwm cov qauv, uas muaj qhov sib txawv ntim qhov ntau thiab tsawg thiab ultrafiltration raj, nyob ntawm seb qhov hnyav molecular ntawm lub hom phiaj protein, lub ntim ua ntej concentration thiab lub hom phiaj ntim ntawm concentration. Reusable.
1. Xaiv ib lub raj ultrafiltration uas tsim nyog, feem ntau xav txog MWCO thiab cov ntim ntim. Feem ntau, qhov hnyav molecular yuav tsum tsis txhob ntau tshaj 1/3 ntawm qhov hnyav molecular ntawm lub hom phiaj protein. Yog tias qhov hnyav molecular ntawm lub hom phiaj protein yog kwv yees li 10 kd, ib lub raj ultrafiltration nrog ib qho hnyav molecular ntawm 3 KD tuaj yeem siv.
2. Cov khoom siv tshiab ultrafiltration yog qhuav. Ntxiv milliq dej ua ntej siv. Cov dej dhau los ntawm daim nyias nyias thiab precooled nyob rau hauv ib da dej dej khov los yog lub tub yees rau ob peb feeb. Tom qab ntawd ncuav tawm cov dej kom ntxiv cov kua protein. Tus nqi ntawm cov kua protein yuav tsum tsis pub tshaj cov kab dawb nyob rau sab saum toj ntawm lub raj kuaj. Kev ua haujlwm yooj yim. Ua ntej ntxiv cov tshuaj protein, lub raj ultrafiltration yuav tsum tau muab tso rau hauv cov dej khov ua ntej txias.
3. Sib npaug. 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 (precooling lub centrifuge rau 4 degrees). Cov kev taw qhia ntawm lub diaphragm thiab lub rotating ncej tuaj yeem hloov kho raws li cov lus qhia (nyob rau hauv cov ntaub ntawv ntawm lub kaum sab xis centrifuge, lub diaphragm yog perpendicular rau lub ncej). Hauv kev siv tiag tiag, qhov dav dav dav yog qis dua li tau teev tseg hauv phau ntawv qhia, uas yuav ua rau lub neej ntev ntawm cov yeeb nkab centrifugal.
4. thaum concentrated mus rau qhov seem 1ml, [noj 50ul tsev neeg Bradford tov, ntxiv 10ul kua los saib seb nws tig xiav, thiab txiav txim seb lub ultrafiltration raj xau cov protein. Yog hais tias lub raj xau, ncuav nws mus rau hauv txheej sab saud thiab tom qab ntawd ntws rov qab mus rau lub raj tshiab kom pib ultrafiltration. Txhawm rau txiav txim siab seb lub raj xa dej puas, thov 5mg 10min centrifuge 10min, tom qab ntawd hla dhau, khiav cov protein gel lossis Bradford] [qhov ntsuas ntxhib]. Txuas ntxiv ntxiv cov tshuaj protein ntxiv kom mloog zoo (ua haujlwm ntawm cov dej khov kom tsis txhob muaj cua sov) kom txog thaum tag nrho cov ntsiab lus ntxiv ntxiv. Thaum lub sij hawm centrifugation, ua tib zoo saib seb cov protein los nag tshwm sim thiab ua rau lub raj thaiv. Yog tias nag lossis daus tshwm sim, thov txiav txim seb qhov ua rau nag lossis daus, seb cov protein ntau npaum li cas los yog qhov tsis tsim nyog; Cov qub tuaj yeem daws tau los ntawm kev siv ntau lub raj ultrafiltration tib lub sijhawm kom txo cov concentration, thaum lub sijhawm tom kawg tuaj yeem hloov pauv cov buffers sib txawv kom txog rau thaum cov protein precipitates.
5. Cov kauj ruam dhau los yog siv los mloog cov protein. Yog hais tias qhov tsis yog yuav tsum tau hloov, thaum tag nrho cov protein tov yog concentrated txog 1ml, maj mam ntxiv ib tug tshiab tsis (ultrafiltration los ntawm 0.22um ultrafiltration membrane), thiab ces tsis tu ncua mloog mus txog 1ml rau peb zaug, yog li ntawd. tias qhov kawg concentration nyob ntawm qhov xav tau cov protein ntau, uas feem ntau tsis ntau tshaj 500ul lossis tsawg dua 200ul. Raws li qhov ntim ntawm qhov tsawg kawg nkaus 10 zaug txhua lub sijhawm, nws yog ntau tshaj 1000 npaug ntawm peb zaug, uas tuaj yeem ua tiav lub hom phiaj ntawm kev hloov pauv tsis.
6. Tshem tawm cov protein ntau concentrated. Ua haujlwm ntawm dej khov. Siv lub taub hau daj daj (200ul), maj mam ntxig lub taub hau ntawm ntug, ces maj mam tshuab thiab sib tov cov kua protein. Ceev faj tsis txhob kov lub ultrafiltration membrane. Nias cov tshuaj concentrated rau 200ul txhua zaus kom txog thaum nws nqus tag. Nws tsis yog qhov tsim nyog los rho tawm cov tshuaj concentrated kawg nyob rau hauv qab ntawm lub raj tshuaj ntsuam xyuas, txwv tsis pub nws yuav nyuaj dhau thiab cov ultrafiltration membrane yuav puas. Thaum kawg, milliq dej yog ntxiv rau lub raj ultrafiltration yam tsis tau dhau los ntawm ultrafiltration membrane los tiv thaiv dej poob thiab ziab ntawm daim nyias nyias.
7. Cov hauv qab no yog cov kauj ruam rau kev kho lub raj ultrafiltration thiab rov siv lub raj ultrafiltration.
8. Ncuav dej rau hauv lub raj ultrafiltration thiab maj mam yaug nrog dej milliq rau ob peb zaug. [yog tias muaj cov kua dej pom tseeb hauv qab ntawm lub raj ntsuas, koj tuaj yeem ntxiv dej ua ntej thiab tom qab ntawd tshuab nws nrog lub pipette. Ceev faj tsis txhob kov daim nyias nyias. Cov pob zeb poob qis thiab khiav tawm mus. Koj tsis tuaj yeem yaug nws nrog dej kais dej. Tom qab ntxiv 0.2m NaOH tov, muab tso rau ntawm chav tsev kub li 20 feeb, thiab sib npaug ntawm ultrafiltration raj lub sij hawm no. Centrifuge rau lwm 10 feeb. Ncuav cov tshuaj NaOH uas tseem tshuav thiab muab tso rau hauv cov paj rwb tub ntxhais hauv lub beaker (1 lossis 2L) Milliq dej, muab tso rau ob peb teev, thiab tom qab ntawd hloov nrog dej tshiab, tso rau ob peb teev kom tsis tu ncua dilute concentration ntawm NaOH. Ntxuav lub 50ml xeem raj thiab npog nrog kais dej, thiab ntxuav lub puab phab ntsa nrog milliq dej.
9. Tshem tawm cov tub ntxhais ntsuas cov tub ntxhais rau hauv dej, ntxiv rau cov dej puv milliq, thiab tom qab ntawd sau lub raj 50ml nrog dej milliq. Maj mam muab cov tub ntxhais rau hauv 50ml.
centrifuge raj, ntws thiab faib dej, ces npog thiab khaws cia ntawm 4 degrees kom txog thaum siv tom ntej. Feem ntau hais lus, raws li cov kauj ruam saum toj no thiab kev ceev faj, txhua lub raj yuav tsis tawg hauv peb lossis plaub xyoos.
