AssemblyoftheUnitforStandardTransfers(Wearglovesforthisproceduretoavoidcontaminationofmembranes.)

1.Removethesafetycoverandthestainlesssteelcathodeassembly.

2.Placethepre-soakedsheetoffilterpaperontotheplatinumanode.Rollapipetortesttubeoverthesurfaceofthefilterpaper(likearollingpin)toexcludeallairbubbles.Ifthinfilterpaperisused,repeatwithtwomoresheetsofbuffer-soakedfilterpaper.

3.Placethepre-wettedblottingmediaontopofthefilterpaper.Rolloutallairbubbles.

4.Carefullyplacetheequilibratedgelontopofthetransfermembrane,aligningthegelonthecenterofthemembrane.Transferwillbeincompleteifanyportionofthegelisoutsidetheblottingmedia.Rolloutallairbubbles.

5.Placeanothersheetofpre-soakedfilterpaperontopofthegel,carefullyremovingairbubblesfrombetweenthegelandfilterpaper.Ifthinfilterpaperisused,placethreesheetsontopofthegel,andremovebubblesfrombetweeneachlayer.

6.Ifmorethanonefull-sizegelistobetransferred,placesheetofpre-soakeddialysismembraneontopofthefilterpaperstack.Repeattheprocedurefromstep2.Uptofourminigelscanbetransferredatthesametimebyplacingthemside-by-sideontheanodeplatform.

7.Carefullyplacethecathodeontothestack.Presstoengagethelatcheswiththeguidepostswithoutdisturbingthefilterpaperstack.

8.Placethesafetycoverontheunit.Plugtheunitintothepowersupply.Normaltransferpolarityiscathodetoanode,i.e.,redwiretotheredoutletandblackwiretoblackoutletonthepowersupply.

Caution:Donotreversepolarity.Thiswillresultindamagetothestainlesssteelcathode.

9.Turnonthepowersupply.Transferminigelsfor15-30min.at10-15V.Largegelscanbetransferredfor30min.to1hr.at15-25V.Donotexceed25Vwiththisinstrument.Acurrentlimit(3mA/cm²forlargegels;5.5mA/cm²forminigels)isrecommendedtopreventexcessiveheatingduringtherun.Underthestrongfieldsdevelopedbythisapparatus,transfersmaynotalwaysbequantitative.Acertainquantityofproteinmaybetransferredthroughthemembraneandontothefilterpaperbelow.(i.e.Constantcurrent~15QmMfor2minigels)

10.Followingtransfer,turnthepowersupplyoff,anddisconnecttheunitfromthepowersupply.Removethesafetycoverandthecathodeassembly.Discardthefilterpaper(anddialysismembrane,ifused).ThetransferefficiencycanbemonitoredbystainingthegelwithCoomassieblueR-250proteinstainorwithBio-Rad’sSilverStainKit.Alternatively,prestainedmolecularweightstandardscanbeused,oraportionofthemembranecanbestainedfortotalproteinwithcolloidalgold,BiotinBlotTotalProteinStain,orananionicdyesuchasAmidoBlack.Zeta-ProbemembranecanbestainedwiththeBiotin-BlotTotalProteinStain.

SDSmaybeaddedtoBuffer1toincreaseproteinelutionfromthegel:

48mMTris,39mMglycine,(20%methanol),1.3mMSDS(0.0375%),pH9.2.Dissolve5.82gTrisand2.93gglycine,and0.0375gSDSor3.75mlof10%SDSinddH₂O(add200mlofmethanol);adjustthevolumeto1literwithdiluteddH₂O.

DONOTADDACIDORBASETOADJUSTpH.

TowbintransferbufferforSDS-proteinsusingnitrocellulose(withmethanol)orZeta-Probemembrane(withoutmethanol):

25mMTris,192mMglycine(20%methanol),pH8.3Dissolve3.03gTrisand14.4gglycineinddH²O(add200mlofmethanol);adjustvolumeto1literwithddH²O.

DONOTADDACIDORBASETOADJUSTpH.

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