什么仪器可以检测电路板元件的好坏ZOL问答
来自 : 蚂蚁淘
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:a
MolecularWeightTransferPeriod<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.
免责声明 本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘”。违反上述声明者，本网将追究其相关法律责任。
