GST-PULL-DOWN手册

TechnicalManualMagneGST™Pull-DownSystemINSTRUCTIONSFORUSEOFPRODUCTV8870.PRINTEDINUSA.Revised3/05Part#TM249AF9TM2490305TM249IMPORTANTNOTICEPleasenotethattheMagneGST™Pull-DownSystemnowincludestheMagneGST™CellLysisReagent.Theincorporationofthisnewbufferenhancescelllysisandyieldwithoutadditionalhandlingsteps.Ifyouhaveadditionalquestions,feelfreetocontactPromegaTechnicalServicesbyphoneat800-356-9526orbye-mail:[e-mail]3/21/200510:11AMPage1PromegaCorporation·2800WoodsHollowRoad·Madison,WI53711-5399USATollFreeinUSA800-356-9526·Phone[phone]·Fax[phone]·:SynthesisofPreyProtein.....................................................................6C.Phase2:ImmobilizationofBaitProtein............................................................6D.Phase3:CaptureofPreyProtein.......................................................................7IV.MagneGST™Pull-DownSystemProtocol..................................................7A.Phase1:ExpressionofthePreyProteinintheTNT®T7QuickCoupledTranscription/TranslationReaction..................................................................8B.Phase2:ImmobilizationofGST-FusionProteinsontoMagneGST™Particles..................................................................................................................9C.Phase3:Capture,WashandAnalysisofPreyProtein.................................12V.Troubleshooting...............................................................................................14VI.CompositionofBuffersandSolutions.......................................................15VII.References.........................................................................................................16VIII.RelatedProducts..............................................................................................16I.DescriptionManyproteinsfunctioninassociationwithpartnerproteinsorascomponentsoflargemultiproteincomplexes.Understandingtheseproteininteractionsiscriticaltoourunderstandingofbiologicalpathwaysandcellularfunction.Theyeasttwo-hybridsystemiscommonlyusedtodetermineprotein-proteininteractionsbutgeneratesmanyfalsepositives,requiringconfirmationofresultsbyanothermethod.GSTpull-down(1)isbecominganimportanttoolforvalidationofsuspectedprotein-proteininteractionsorfordiscoveringnovelproteininteractions.GSTpull-downusesaGST-fusionprotein(bait)boundtoglutathione(GSH)-coupledparticlestoaffinitypurifyanyproteins(prey)thatinteractwiththebaitfromapoolofproteinsinsolution(2).Baitandpreyproteinscanbeobtainedfrommultiplesourcesincludingcelllysates,purifiedproteins,expressionsystemsandinvitrotranscription/translationsystems.MagneGST™[e-mail]3/21/200510:11AMPage1TheMagneGST™Pull-DownSystem(a–g)providesGSH-linkedmagneticparticlesthatallowsimpleimmobilizationofGST-fusionbaitproteinsfrombacteriallysatesandaninvitrotranscription/translationsystemforexpressionofpreyproteins.PreyproteinsareisolatedusingtheappropriatebaitproteinattachedtotheMagneGST™(GSH)Particles(a).TheMagneGST™Pull-DownSystemprotocolcanbeusedfordetectionofinteractionsbetweenGST-fusionproteinsexpressedinbacteriallysatesandpreyproteinsexpressedintheTNT®System.Theprotocolisdividedintothreephases:1)thepreyproteinisexpressedinthetranscription/translationreaction;2)theE.coli-expressedGST-fusion(bait)proteinisimmobilizedontotheMagneGST™Particles;and3)thepreyproteinisaddedtotheMagneGST™Particlescarryingbaitandcapturedthroughbait-preyinteraction.Phases1and2areperformedsimultaneously(Figure1).Thetranscription/translationcomponentoftheMagneGST™Pull-DownSystemistheTNT®T7QuickMasterMix,whichallowstheconvenientsingle-tubecoupledtranscription/translationofgenescloneddownstreamfromT7RNApolymerasepromoters.TheTNT®System(b–d,h,i)iscompatiblewithcircular(plasmid)orlinear(plasmidorPCRproduct)templates.Forimmobilizationandpurificationofbaitproteins,theMagneGST™Pull-DownSystemprovidestheMagneGST™CellLysisReagenttolysebacterialcellsandMagneGST™ParticlesforbindingGST-fusionbaitproteins.PreyproteinsynthesizedintheTNT®T7QuickCoupledTranscription/TranslationSystemreactioniscapturedusingbaitprotein(GST-fusionprotein)immobilizedonMagneGST™Particles.Nonspecificallyboundproteinsarethenwashedawayandthepreyproteinisanalyzed