western - the nystul lab

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1、WesternBlot-ElectrophoresisIntroduction:ThisisthestandardlabprotocolforsettingupandrunningaWestern.ItwasadaptedfromtheInvitrogenprotocolandisdesignedforusewithInvitrogenproducts.ElectrophoresisMaterials:·Proteinsamples·XCellSureLockMini-Cell·Gel:NuPAGEBis_Trisgel·Loadingbuffer

2、:NuPAGELDSSampleBuffer(4x)·Runningbuffer:NuPAGEMOPSSDSRunningBuffer(20X)·ddH2O·NuPAGESampleReducingAgent(10X)·NuPAGEAntioxidant·Proteinladder·70degreewaterbathProcedure–SamplePreparation:1.Heatwaterbathto70degrees.2.Thawproteinsamplesandproteinladderinice.3.Preparerunningbuffe

3、r(900ml):-45mlSDSRunningBuffer+855mlddH2Oo600mlforlowerbufferchambero200mlforupperbufferchamber(add500ulAntioxidantwithin30minutesofloading)4.Prepareproteinsamples:-Protein:10pairsofovaries-Samplebuffer:25ul-Reducingagent:10ul-ddH2O:15ul-total:50ulà5ul/pair(whichisgenerallyago

4、odamounttoload)5.Heatproteinsamplesat70degreesfor10minutes.Removefrombathandplaceoniceimmediately.Notes:·Theconcentrationoftheproteinsamplecanchangebaseduponthegel’swellsizeandthenumberofovariesyouwanttorunperlane.Keeptheratioofbuffertoreducingagentthesame,andbesuretonotewhatc

5、oncentrationyouareusing.Procedure–Electrophoresis:1.Cutopenthegelcassettepouchwithscissorsandremovecassette.2.Drainawaythegelpackagingbuffer.3.RemovethegelcassettefromthepouchandrinsewithddH2O.*Note:Alwayshandlethecassettebyitsedgesonly.4.Peeloffthetapecoveringtheslotontheback

6、ofthegelcassette.5.Inonefluidmotion,pullthecomboutofthecassette.Thisexposesthegelloadingwells.6.Useapipettetogentlywashthecassettewellswith1Xrunningbuffer.Invertthegelandshaketoremovebuffer.Repeattwice.Fillthesamplewellswithrunningbuffer.Note:Besuretodisplaceallairbubblesfromt

7、hecassettewellsastheywillaffectsamplerunning.7.LowertheBufferCoreintotheLowerBufferChambersothatthenegativeelectrodefitsintotheopeninginthegoldplateontheLowerBufferChamberasshowninthefigure.8.InserttheGelTensionWedgeintotheXCellSureLock™behindthebuffercore.MakesuretheGelTensio

8、nWedgeisinitsunlockedposition,allowingthewedgetoslipeasilyint