Momwe mungagwiritsire ntchito guluu wolekanitsa?- Gawo 1

Momwe mungagwiritsire ntchito guluu wolekanitsa?

Kutumiza kwa nembanemba waku Western blot

Pansi pa zomwe zikuchitika pano, mapuloteni amasamutsidwa kuchoka ku gel osakaniza kupita ku chonyamulira cholimba (membrane).

Kusankhidwa kwa Membrane: Zomwe zimagwiritsidwa ntchito nthawi zambiri zolimba-gawo mu imprinting zikuphatikizapo NC nembanemba, DBM, DDT, nayiloni nembanemba, PVDF, etc. Timasankha PVDF (polyvinylidene fluoride), amene ali bwino mapuloteni adsorption, mphamvu thupi ndi bwino zogwirizana mankhwala.Pali mitundu iwiri: immobilon-p (0.45um) ndi immobilon PSQ (0.2um ya MW <20kDa).

Njira ya semi dry

Ndiko kuti, kuphatikiza kwa gel interlayer kumayikidwa pakati pa pepala losefera ndi chotengera chosinthira, ndipo chapano chimayendetsedwa kudzera pa buffer yomwe imayikidwa pa pepala losefera kuti ikwaniritse kusintha.Chifukwa chapano chimachita mwachindunji pagulu la filimuyo, kusamutsidwa kumakhala kovutirapo, koma nthawi yosinthira ndi yaifupi ndipo magwiridwe antchito ndi apamwamba.

(1) kusankha mikhalidwe yoyesera

Pakalipano ndi 1ma-2ma / cm2, ndipo nthawi zambiri timagwiritsa ntchito 100mA / nembanemba.Nthawi yosinthira imatha kusankhidwa molingana ndi kukula kwa molekyulu ya protein yomwe mukufuna komanso kuchuluka kwa gel, ndipo imatha kusinthidwa moyenera malinga ndi momwe zilili.

Kukula kwa mamolekyulu a protein (kDa), ndende ya gel ndi nthawi yosinthira

80---140 8% 1.5-2.0

25---80 10% 1.5

15-40 12% 0.75

<20 15% 0.5

(2) ntchito yoyesera

(1) Kukonzekera kwa pepala losefera ndi nembanemba (kukonzekera kudzayamba mphindi 20 isanafike electrophoresis).

A. Onani ngati pali mabafa okwanira osamutsira, omwe sanakonzekere nthawi yomweyo.

B. Onani ngati pali pepala losefera ndi nembanemba ya kukula koyenera.

C. Miwiritsani nembanemba mu methanol kwa mphindi 1-2.Ndiyeno anasamutsidwa kulanda bafa.

D. Zilowerereni pepala loyenera lomatira losefera ndi pepala losefera la membrane muzotchingira zotengera motsatana.

Kodi nembanemba ya PDVF iyenera kulowa mu methanol mpaka liti ikalowa mu buffer?

PVDF ndi hydrophobic, ndipo ndizovuta kuti zilowerere mu buffer kusamutsa.Pambuyo pa mankhwala a methanol, zimakhala zosavuta kuti zilowerere.Kukonzekera kwa PVDF ndikunyowetsa ndikuyambitsa ndi methanol, ndikutsuka kawiri ndi madzi osungunuka mutatha kuviika bwino.Cholinga chogwiritsira ntchito thovu la methanol ndikuyambitsa magulu omwe ali ndi mphamvu zabwino pa membrane ya PVDF ndikupangitsa kuti zikhale zosavuta kumanga ndi mapuloteni opanda pake.

Nthawi zambiri anthu amagwiritsa ntchito kwa mphindi 5-20.Itha kuviikidwa nthawi yomweyo ndikutenga guluu, ndipo akuti zitenga pafupifupi mphindi 5.Zotsatira zake sizoyipa.M'mbuyomu, abwenzi ena a pa intaneti adalemba kuti nthawi yokonzekera inalibe kanthu, koma chinsinsi chinali khalidwe la filimuyo.Ndizowona.Malingana ngati nembanembayo yanyowa kwathunthu, iyenera kukhala bwino.

Mafotokozedwe a Hybond a PVDF amawerengedwa motere: "pre wet the membrane in 100% metanol (10 seconds)".Kumvetsetsa kwanga ndikuti ndikwabwino kuviika kwa masekondi 10.M'malo mwake, ndi bwino kuti zilowerere kwa masekondi 10 kapena mphindi 10.

(2) Kusamutsa

A. Ikani pepala losefera m'munsi pa chida chosinthira magetsi.Nthawi zambiri zigawo zitatu zimagwiritsidwa ntchito.

B. Yalani nembanemba pa pepala losefera motsutsana ndi nembanemba, samalani kuti pasakhale thovu pakati pa nembanemba ndi pepala losefera, ndipo tsanulirani zotchingira zosinthira pa nembanemba kuti nembanemba ikhale yonyowa.

C. Pewani guluu, chotsani gel osakaniza ndikusuntha mosamala pafilimuyo.

D. Dulani kumtunda kumanzere ngodya ya filimuyo ndi kuika guluu udindo pa filimuyo ndi pensulo.

E. Phimbani pepala losefera ndi guluu paguluu.Thirani bafa ina yosinthira ndikuyika zidutswa ziwiri za pepala losefera.

F. Ikani chida chosinthira magetsi ndikusankha zomwe mukufuna komanso nthawi malinga ndi zosowa.

G. Panthawi yosinthira, kusintha kwa magetsi kudzawonedwa nthawi iliyonse, ndipo zolakwika zilizonse zidzasinthidwa nthawi.