Bis-Tris-gel配方

TomakeBis-Trisgelwith1.5mmcombs8%12%Separatinggel1gel2gels4gels6gels8gels1gels2gels4gelsMilli-Qwater3.7ml7.4ml14.8ml22.2ml29.6ml2.7ml5.4ml10.8ml3.5×Bis-Trisgelbuffer2.3ml4.6ml9.2ml13.8ml18.4ml2.3ml4.6ml9.2ml32%Acrylamide-Bisgel2.0ml4.0ml8.0ml12ml16ml3.0ml6.0ml12ml10%APS75ul150ul300ul450ul600ul50ul100ul200ulTEMED5ul10ul20ul30ul40ul3ul6ul12ulTotal8ml16ml32ml48ml64ml8ml16ml32mlStackinggel1gel2gels4gels6gels8gelsMilli-Qwater1.76ml3.52ml7.04ml10.56ml14.08ml3.5×Bis-Trisgelbuffer0.86ml1.72ml3.44ml5.16ml6.88ml32%Acrylamide-Bisgel0.38ml0.76ml1.52ml2.28ml3.04ml10%APS20ul40ul80ul120ul160ulTEMED4ul8ul16ul24ul32ulTotal3ml6ml12ml18ml24mlBis-Trisgelbufferprotocol1.Make32%Acrylamide/Bis-acrylamide(30:2)solution30gAcrylamide2gBis-acrylamide50mlMilli-QwaterLetAcrylamide/Bis-acrylamidedissolveinwater,thenaddwaterupto100mlsolution.Wrapwithaluminumfoil,keepat4℃.2.20×lowMWrunningbuffer(1L)MES1000mM195.6gTris1000mM121.12gEDTA20mM7.44g(EDTA-2Na-2H2O)SDS2%20gAdd600mldeionizedH2Otodissolvefirst,thenwaterupto1L.NoneedtoadjustpH.Addsodiumbisulfateto5mM(addfreshbeforerun)forma1Mstock.3.5×highMWrunningbuffer(1L)----forseparating20kDproteinsMOPS250mM52.32gTris250mM30.28gEDTA5mM1.86g(sodiumEDTA)SDS0.5%5gAdd600mldeionizedH2Otodissolvefirst,thenwaterupto1L.NoneedtoadjustpH.Addsodiumbisulfateto5mM(addfreshbeforerun)forma1Mstock.4.200×runningbufferreducingagent500ml1Msodiumbisulfateaddtorunningbufferat5mMfinalconcentration52gsodiumbisulfatedissolvein500mldeionizedwater.5.3.5×gelbuffer(100ml)1.25MBis-Tris(adjustpHto6.5-6.8withHCl)---Thatis:Bis-Tris26.16g,dissolvein100mlMilli-Qwater(4.5-5ml浓HCl).Note:bis-TrisisBis(2-hydroxyethyl)aminotris(hydroxymethyl)methane(e.g.Sigmacatalog#B7535)6.WBtrans-buffer-1X(8L)Tris-base30.25gGlycine150g