Western Blotting Using the SemiPhor Semi-Dry Transfer Unit-蚂蚁淘商城

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Western Blotting Using the SemiPhor Semi-Dry Transfer Unit

2025-06-16

WesternBlottingUsingtheSemiPhorSemi-DryTransferUnit

Removethestackinggelfromthegel.Measurethegelandrecorditssize.Donotpre-soakthegel.Donotcutacorneroffthegel,asthismayallowashortcircuitandinefficienttransfer.BlotthegelassoonaspossIBLeafterelectrophoresistoavoiddiffusionofthesamplesthroughthegel.

Cutpiecesofblotterpapertothesizeofthegel.Youwillneedfourpieces,plusoneforeachgeltransferred.Itisimportantthattheblotterpaper(andthemembrane)notbelargerthanthegel.Largerpiecesmaymakecontactaroundthegel,andallowthecurrentanalternateroute,therebymakingtransferinefficient.

Wearingglovesrinsedofpowder,cutapieceofnylonmembranetothesizeofthegel.Markthelowerright-handcornertoallowfororientation.Pre-wetthemembraneintransferbufferfor2to5minutes.

PlaceamylarmaskinthebottomoftheSemiPhorunit.Themaskshouldhaveacentral,squareopeningcutinitthatis2mmsmallerthanthegelinbothlengthandheight.

Inadishoftransferbuffer,saturatetwopiecesofblotterpapercutinstep2.Placetheseontopofthemylarmask,centeringthembyfirstplacingthecenterofthepaperdownfirst,androllingtheedgesout.Thefilterpapershouldcoverthecut-outinthemaskandslightlyoverlapitonallsides.

Constructthefirsttransfersandwichontopofthepreviously-placedblotterpaperbyplacingthemembrane,thenthegel,thenanadditionalpieceofbuffer-soakedblotterpaperonthestack.Uptofivegelsofthesamesizemaybetransferredsimultaneously.Ifthisisthecase,placeasheetofcellophane(cutslightlysmallerthanthegel,itwillswellwhenwet)ontopofthefirsttransfersandwich,andconstructthenextoneontop(membrane,gel,blotterpaper;seediagram).

Stackthecomponentsneatly,withedgesparallel.Aseachlayerisplaceduponthestack,makesurenoairbubblesaretrapped.Sweepawetted,glovedfingeroverthelayer,orrollaPipetteortesttubeoverittoremovebubbles.Proteinsonthesurfaceofthegelwillbindtothemembraneassoonascontactismade.Themembranemustthereforebepositionedcorrectlythefirsttime.Donottrytoadjust.
Placetwoadditionalpiecesofbuffer-soakedblotterpaperontopofthestack.Placethecoverontheunit(itfitsonlyoneway).Holdthecoverlevelandslideitgentlydownontothestack.
- Donotremovethecoveruntilaftertheblotiscompleted.Partofthestackmaysticktothecoverorotherwisebeknockedoutofalignment.
- Whentransferringmultiplegels,itmaybedesirabletoweighdownthelidinordertoensureevencontactwiththestack.Inthiscase,placeaweight(upto1kg)onthecover.Toomuchweightwillcompressthestackandhindertransfer.
- Connecttheunittoasuitablepowersupply.Turnthepowersupplytozerobeforeturningon.Turnonthepowersupplyandsettoapproximately0.8mA/cm2ofgel.8mmx7mmmini-gelsmayberunat100mAconstantcurrent.Largergelsmustbelimitedto0.8mA/cm2toavoidexcessiveheatbuildup.Allowthetransfertoproceedforthelengthoftimespecifiedinthetable.
  AssemblyoftheTransferStack
  Approximatedurationoftransferrunsaccordingtomolecularweight:aMolecularWeightTransferPeriod<20,00015minutes20,000-80,000030minutes>80,00045minutesa8mmx7mmmini-gelsat100mA.TransferBuffer(500ml,pH8.3)glycine1.450g(39mM)Trisbase2.900g(48mM)SDS0.185g(0.037%)methanol100.00ml(20%)Combinereagentsinafinalvolumeof500mldH2O.UseelectrophoresisgradeSDS.Themethanolisnotnecessarywhenusingpositively-chargedmembranes.