DNA-pull-down

DNAPull-downProtocolGroupMeeting06-21-2005+DynabeadsstreptavidinBioinylatedDNAfragmentDNAcontaininggenepromotersequenceDynabeadsboundDNAfragmentAddcellextracttodynabeads,incubationMagneticseparation,removenon-DNAbindingproteinsIsolateDNAbindingproteinsNon-DNAsequencespecificproteinsIdentificationandcharacterizationEluteDNAsequencespecificproteinDNAPull-downProtocolBeadsustaintemperatureDynaBeadsendurabletemperatureheating60oC85oC70oC75oC80oC90oCstreptavidin30minIncubationTemperature70oC55oC10kDa-15k-20k-25k-37k-50k-75k-100k-150k-250k-DNAimmobilizationDNA–cellextractIncubation70oC/55oCWashEluteproteinsRecombinantLrpASaltconcentrationforincubationABABABABABABWash1stWash2ndWash3rdDNasedigestFinaleluteA0.1MKClvsB0.5MKClRecombinantLrpASaltconcentrationinwashingA0.4MB0.2MDNaseeluteKClBALrpAThermosomesinglesubunitWashLrpADifferentproteinamount281610(kDa)-15-20-25-37-50-75-100-150-LrpACellextract(mg/mL)IdentificationofnaturalLrpAfromPfcellextract300bppromoterDNAincubatewithPfcellextract,NorecombinantLrpAaddedNaturalLrpAwasidentifiedNaturalLrpAidentificationlrpAamysipAlrpAamysipALrpA-10(kDa)-25-15-206mg/mlproteinsOthers•Proteinsfractionationmethods–FragmentDNA–PolydI-dC,SalmonDNA,CalfDNA,HerringDNA•Differentmethods–Cellextract+promoterDNAcompetitorDNA–Cellextract+promoterDNA+competitorDNA–Cellextract+competitorpromoterDNA176bpATG528bpF1F2F3R1R2R3DNAimmobilizationonDynaBeads•DNAlength–300basepairs•Buffer-2xB&Wbuffer–10mMTris,1mMEDTA,2.0MNaCl,adjustthepHto~7.0byHCl•Steps1.Take50ulbeads(forexample)andwashwith2xB&Wbuffertwice,100ul/time.(ifusingmorebeads,scaleupeverythingelseinthefollowingsteps)2.ImmobilizeenoughDNA(about0.39pm300bpDNA/ulbeads)onbeadsin1xB&Wbuffer,incubatefor15mins(1kbDNA)underroomtemperature(RT).Keepthebeadsshakinggentlytopreventprecipitationinsolution.3.Twicequickwashforbeads-DNAwith1xB&Wbuffer,100ul/timeProteinandDNAincubation•Buffer–incubationbuffer–50mMTris,1mMEDTA,100mMKCl,adjustpHto~7.0byHCl,5%Glycerol,0.1%TritonX100–Addfreshlymade100mMDTTtoreactionsolutiontomakefinalconcentrationofDTTat1mM.•Steps1.WashBeads-DNAwithincubationbuffer100ul/timefor3times.2.IncubateBeads-DNAwith2.5-5mg/mlofcellextract100ulfor30minat70oConaheater.3.Gentlyshakeandsuspendthebeadsevery2-3minstoavoidbeadsprecipitation.Afterincubation,removethesupernatant(extracellextract).Removenon-specificDNAbindingproteins•Buffer–Incubationbuffer•Steps–3timesofquickwashforbeadsboundDNA-proteincomplexwith100ulwashingbuffer@RTRemovenon-DNA-bindingproteinsbyremovingsupernatantAddwashingbuffer3xRTProteineluting•Buffer–DNasereactionbuffer–Laemmlibuffer•Methods–(Optional,iffollowedbyproteinelectrophonesis)TurboDNasedigestofBeads-DNAcomplexfor30minat37oC.40uldigestionsolutioncomposedby4ulDNase,4ul10xdigestionbuffer,32ulnanopurewater.Shakethetubefrequentlytoavoidsedimentofbeadsinsolution.–Add50ul1xLaemmlibuffer,andheatat50oCfor10mintoelutemostproteins.Takesupernatantandforgelanalysis.–HeattheBeadsleftinthemicrotubeat100oCfor5minsin1xLaemmlibuffer50ul.(optional,probablynothingleftonbeads).