emsa 中文操作说明

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1、ProcedureforElectrophoreticMobilityShiftAssay(EMSA)Thiskithasbeenoptimizedforusewithpolyacrylamidemini(8×8×0.1cm)gels.Forlargergels,adjustelectrophoresisconditionsanddetectionreagentvolumesaccordingly.A.PlanBindingReactions•UnderstandingtheControlEBNASystemIncludeacompletesetofthreer

2、eactionseachtimeanEMSAisperformed.ThesereactionsandexpectedresultsfortheControlEpstein-Barrnuclearantigen(EBNA)System,whichisincludedwiththekit,aredescribedinTable1.Table1.Descriptionofcontrolreactionsandexpectedresults.ReactionContentsofReactionDescriptionResult#1Biotin-EBNAControlD

3、NANoproteinextractforDNAtobind;therefore,noshiftisobserved.Establishesthepositionofanunshiftedprobeband.#2Biotin-EBNAControlDNA+EBNAextractContainssufficienttargetproteintoeffectbindingandshiftoftheBiotin-EBNADNA.Shiftdetectedbycomparisontobandpositionin#1#3Biotin-EBNAControlDNA+EBNA

4、extract+200-foldmolarexcessofunlabeledEBNADNADemonstratesthatthesignalshiftobservedin#2canbepreventedbycompetitionfromexcessnon-labeledDNA,i.e.,theshiftresultsfromspecificprotein:DNAinteraction.TheControlEBNASystemresultsreportedinTable1weregeneratedusingbindingreactionspreparedaccor

5、dingtoTable2.Each20µlbindingreactioncontains20fmolofBiotin-EBNAControlDNA.Reactionswereelectrophoresed,transferredanddetectedaccordingtothestepsinSectionsB-Gofthisprotocol.Ifthekitisbeingusedforthefirsttime,performtheControlEBNASystemreactionstoverifythatthekitcomponentsandoverallpro

6、cedureareworkingproperly.Table2.BindingreactionsforControlEBNASystem.ComponentFinalAmountControlReactions#1#2#3UltrapureWater----12μL11μL9μL10XBindingBuffer(20148A)1X2μL2μL2μL50%Glycerol(20148F)2.5%1μL1μL1μL100mMMgCl2(20148I)5mM1μL1μL1μL1µg/μLPoly(dI•dC)(20148E)50ng/µL1μL1μL1μL1%NP-4

7、0(20148G)0.05%1μL1μL1μLUnlabeledEBNADNA(20148C)4pmol--------2μL5/5EBNAExtract(20148D)1Unit----1μL1μLBiotin–EBNAControlDNA(20148B)20fmol2μL2μL2μLTotalVolume----20μL20μL20μLTable3.BindingreactionsfortheTestSystem.ComponentFinalAmountReaction#1#2#3UltrapureWater……10XBindingBuffer(20148A

8、)1X2μL2μL2μL