Nzira yekushandisa sei kuparadzanisa glue?- chikamu 1

Nzira yekushandisa sei kuparadzanisa glue?

Western blot membrane transfer

Pasi pechiito chezvino, puroteni inotamiswa kubva kujeri kuenda kune yakasimba mutakuri (membrane).

Kusarudzwa kweMembrane: iyo inowanzoshandiswa yakasimba-chikamu zvinhu mu imprinting zvinosanganisira NC membrane, DBM, DDT, nylon membrane, PVDF, etc. Tinosarudza PVDF (polyvinylidene fluoride), iyo ine nani mapuroteni adsorption, simba remuviri uye kuwirirana kwemakemikari zviri nani.Pane zvirevo zviviri: immobilon-p (0.45um) uye immobilon PSQ (0.2um yeMW <20kDa).

A semi dry method

Ndokunge, iyo gel interlayer musanganiswa inoiswa pakati pebepa resefa neiyo yekuchinjisa buffer, uye yazvino inoitwa kuburikidza nebuffer adsorbed pabepa resefa kuti uwane mhedzisiro yekuchinjisa.Nemhaka yekuti ikozvino inobata zvakananga pane firimu glue, mamiriro ekutamisa ane hutsinye, asi nguva yekufambisa ipfupi uye kushanda kwakakwirira.

(1) kusarudzwa kwemamiriro ekuedzwa

Ikozvino ndeye 1ma-2ma / cm2, uye isu tinowanzo shandisa 100mA / membrane.Nguva yekufambisa inogona kusarudzwa maererano nehukuru hwechinangwa cheprotein molecule uye gel concentration, uye inogona kugadziriswa zvakakodzera maererano nemamiriro chaiwo.

Target protein molecular size (kDa), gel concentration uye nguva yekufambisa

80---140 8% 1.5-2.0

25---80 10% 1.5

15—40 12% 0.75

<20 15% 0.5

(2) kuedza kushanda

(1) Kugadzirira kwepepa resefa uye membrane (kugadzirira kuchatangwa maminitsi makumi maviri pamberi pe electrophoresis).

A. Tarisa kana paine mabhafa ekutamisa akakwana, asina kugadzirwa nekukasika.

B. Tarisa uone kana paine sefa bepa uye membrane yehukuru hwakakodzera.

C. Nyura membrane mumethanol kweinenge 1-2 maminetsi.Uye wozoendeswa kune yekuchinjisa buffer.

D. Nyura bepa rakakodzera rekunamira sefa uye membrane sefa bepa mubhafa yekutamisa zvichiteerana.

Iyo PDVF membrane inofanirwa kunyura mumethanol kwenguva yakareba sei kana ichipinda mubhafa yekufambisa?

PVDF ndeye hydrophobic, uye zvakaoma kunyura mubhafa yekufambisa.Mushure mekurapa methanol, zviri nyore kunyura.Iyo pretreatment yePVDF ndeyekunyorovesa nekuimisa nemethanol, uye woigeza kaviri nemvura yakadirwa mushure mekunyatsonyorova.Chinangwa chekushandisa methanol bubbles ndechekuita kuti mapoka anochajiswa zvakanaka paPVDF membrane uye kuita kuti zvive nyore kusunga nemapuroteni asina kunaka.

Vanhu vanowanzoshandisa iyo kwemaminitsi 5-20.Inogona kunyoroveswa panguva imwe chete nekutora glue, uye inofungidzirwa kuti inotora anenge maminitsi mashanu.Mhedzisiro yacho haina kuipa.Munguva yakapfuura, dzimwe shamwari dzewebhu dzakatumira kuti nguva yekugadzirisa yakanga isina basa, asi kiyi yaiva yemhando yefirimu.Ichokwadi.Chero bedzi iyo membrane yakanyura zvachose, inofanira kunge yakanaka.

Hybond's PVDF yakatarwa inoverenga seizvi: "pre wet the membrane mu 100% metanol (10 seconds)".Kunzwisisa kwangu ndekwekuti zvakanaka kunyura kweanenge masekonzi gumi.Muchokwadi, zvakanaka kunyura kwemasekonzi gumi kana maminitsi gumi.

(2) Kutamisa

A. Isa bepa rezasi resefa pane chiridzwa chekufambisa chemagetsi.Kazhinji mitsara mitatu inoshandiswa.

B. Isa membrane pabepa resefa pakatarisana ne membrane, chenjerera kuti pasava nemafuru pakati pe membrane nebepa resefa, uye dururira imwe buffer pa membrane kuti membrane irambe yakanyorova.

C. Bvisa glue, bvisa stack gel uye unyatsoifambisa pafirimu.

D. Cheka chepamusoro kuruboshwe kona yefirimu uye nyora nzvimbo yeglue pafirimu nepenzura.

E. Vhara bepa resefa uchitarisana nenamo panamo.Dururira imwe buffer yekufambisa uye woisa zvidimbu zviviri zvesefa bepa.

F. Isa chimbo chekuendesa magetsi uye sarudza ikozvino inodiwa uye nguva maererano nezvinodiwa.

G. Munguva yekushandura, shanduko yemagetsi ichaonekwa chero nguva, uye chero ipi zvayo isina kukwana ichagadziriswa munguva.