Yuav siv cov kua nplaum sib cais li cas?– part 1

Yuav siv cov kua nplaum sib cais li cas?

Western blot membrane hloov

Nyob rau hauv qhov kev txiav txim ntawm tam sim no, cov protein yog pauv los ntawm gel mus rau cov khoom thauj khoom (membrane).

Membrane xaiv: cov khoom siv feem ntau siv rau hauv kev luam ntawv suav nrog NC daim nyias nyias, DBM, DDT, nylon membrane, PVDF, thiab lwm yam. Peb xaiv PVDF (polyvinylidene fluoride), uas muaj cov protein ntau zoo dua, lub cev muaj zog thiab cov tshuaj zoo sib xws.Muaj ob qhov tshwj xeeb: immobilon-p (0.45um) thiab immobilon PSQ (0.2um rau MW <20kDa).

Ib txoj kev qhuav qhuav

Ntawd yog, cov gel interlayer ua ke yog muab tso rau nruab nrab ntawm cov ntawv lim nrog cov hloov tsis tau, thiab tam sim no yog ua los ntawm cov tsis adsorbed ntawm daim ntawv lim kom ua tiav cov nyhuv hloov.Vim tias tam sim no ua ncaj qha rau ntawm cov nplaum zaj duab xis, cov kev hloov pauv tau hnyav heev, tab sis lub sijhawm hloov pauv luv luv thiab qhov ua tau zoo yog siab.

(1) xaiv qhov kev sim siab

Tam sim no yog 1ma-2ma / cm2, thiab peb feem ntau siv 100mA / daim nyias nyias.Lub sijhawm hloov pauv tuaj yeem raug xaiv raws li qhov loj ntawm lub hom phiaj protein molecule thiab gel concentration, thiab tuaj yeem hloov kho kom haum raws li qhov xwm txheej tiag tiag.

Target protein molecular loj (kDa), gel concentration thiab hloov lub sij hawm

80---140 8% 1.5-2.0

25---80 10% 1.5

15-40 12% 0.75

<20 15% 0.5

(2) kev sim ua haujlwm

(1) Kev npaj cov ntawv lim thiab daim nyias nyias (kev npaj yuav tsum pib 20 feeb ua ntej electrophoresis).

A. Xyuas seb puas muaj cov hloov pauv txaus, uas tsis tau npaj tam sim ntawd.

B. Xyuas seb puas muaj cov ntawv lim thiab daim nyias nyias ntawm qhov loj.

C. Tso cov membrane rau hauv methanol li 1-2 feeb.Thiab ces pauv mus rau qhov hloov tsis tau.

D. Soak cov ntawv nplaum uas tsim nyog thiab cov ntawv lim membrane rau hauv qhov hloov tsis tau raws li.

Ntev npaum li cas PDVF daim nyias nyias yuav tsum tau tsau rau hauv methanol thaum nkag mus rau qhov tsis hloov pauv?

PVDF yog hydrophobic, thiab nws yog ib qho nyuaj rau soak nyob rau hauv lub hloov tsis.Tom qab kev kho methanol, nws yooj yim dua rau soak.Lub pretreatment ntawm PVDF yog tsau thiab qhib nws nrog methanol, thiab ces ntxuav nws ob zaug nrog distilled dej tom qab soaking kom huv si.Lub hom phiaj ntawm kev siv cov npuas methanol yog txhawm rau qhib cov pab pawg tau txais txiaj ntsig zoo ntawm PVDF daim nyias nyias thiab ua kom yooj yim rau khi nrog cov protein tsis zoo.

Cov neeg feem ntau siv nws li 5-20 feeb.Nws tuaj yeem tsau rau tib lub sijhawm thaum noj cov kua nplaum, thiab kwv yees tias nws yuav siv li 5 feeb.Cov nyhuv tsis yog phem.Yav dhau los, qee tus phooj ywg hauv web tau tshaj tawm tias lub sijhawm ua haujlwm tsis muaj teeb meem ntau, tab sis qhov tseem ceeb yog qhov zoo ntawm zaj duab xis.Qhov tseeb tiag.Ntev npaum li daim nyias nyias tau ntub tag, nws yuav tsum tau OK.

Hybond's PVDF specification nyeem raws li hauv qab no: "ua ntej ntub cov membrane hauv 100% metanol (10 vib nas this)".Kuv txoj kev nkag siab yog tias nws yog OK kom ntub dej tsawg kawg 10 vib nas this.Qhov tseeb, nws yog OK rau soak rau 10 vib nas this lossis 10 feeb.

(2) Hloov

A. Tso cov ntawv lim qis rau ntawm lub tshuab hluav taws xob hloov hluav taws xob.Feem ntau peb txheej yog siv.

B. Tso lub membrane rau ntawm daim ntawv lim tawm ntawm daim nyias nyias, ua tib zoo saib kom tsis txhob muaj npuas ntawm daim nyias nyias thiab cov ntawv lim, thiab ncuav ib co hloov tsis mus rau hauv daim nyias nyias kom cov membrane ntub.

C. Tshem tawm cov kua nplaum, tshem tawm cov gel thiab ua tib zoo txav mus rau hauv zaj duab xis.

D. Txiav lub kaum sab laug sab sauv ntawm zaj duab xis thiab kos lub nplaum txoj hauj lwm ntawm zaj duab xis nrog ib tug cwj mem.

E. Npog ib daim ntawv lim tawm tsam cov kua nplaum ntawm cov kua nplaum.Ncuav ib co hloov tsis tau thiab nteg ob daim ntawv lim.

F. Nruab lub ntsuas hluav taws xob hloov hluav taws xob thiab xaiv qhov xav tau tam sim no thiab lub sijhawm raws li qhov xav tau.

G. Thaum lub sijhawm hloov pauv, qhov hloov hluav taws xob yuav tsum raug soj ntsuam txhua lub sijhawm, thiab qhov txawv txav yuav tsum tau hloov kho raws sijhawm.