pull-down技术

DetectionofProtein-ProteinInteractionsUsingtheGSTFusionProteinPull-downTechniqueBacteriallyexpressedglutathioneS-transferase(GST)-fusedproteinsareusedasprobestoperformdirectmeasureofprotein-proteininteractionsandforaffinitypurification.TheGSTFusionproteinpull-downtechnique(Kaelinetal.1991)usestheaffinityofGSTforglutathione-coupledbeadstopurifyinteractingproteinsfromasolutionofnoninteractingproteins.TheGSTfusionproteinprobeisexpressedandpurifiedfrombacteria;Inparallel,acelllysate(whichcanbe35S-labledorunlabled)isprepared;TheGSTfusionproteinprobeandthecelllysatearemixed,inthepresenceofglutathione-agarosebeadsandincubatethemixturetoallowproteinassociationstooccure;Bycentrifugation,collecttheGSTfusionprobeproteinandanyassociatedmolecules;ThecomplexesarewashedandcanbeelutedfromthebeadswithexcessfreeglutathioneorboileddirectlyinSDS-PAGEbuffer;TheproteinsareresolvedbySDS-PAGE,andprocessedbyWesternblotting,autoradiographyorproteinstaining.GSTProteinXGST35S-labledcelllysate(glutathione-sepharosebeads)(glutathione-sepharosebeads)35S-labledcelllysateGSTProteinXGSTInteractat40CMicrofugetocollectcomplexes123autoradiographAnalyzebySDS-PAGELane1.MarkerLane2.GST-proteinXLane3.GSTTheschemaofaGSTpull-downexperimentGSTProteinXTwogeneraluses:Toidentifynovelinteractionsbetweenafusion(orprobe)proteinandunknown(ortarget)proteins;1)Theproteinconcentrations,2)Istheprobeproteinnormallyexpressedinthatparticularcellortissue?3)Isthegoaltocomparedifferenttypesofcellpopulations?Toconfirmsuspectedinteractionsbetweentheprobeproteinandaknownproteins.antibodiestothetargetprotein,or:1)the35S-labeledinvitrotranslatedprotein,orthetargetproteincanbetaggedwithanepitope;2)Cellinculturecanbetransfectedwithaplasmidencodingthetargetproteintoincreasetheabundance;3)Tocontrolthespecificityofbinding,thebestistheinclusionofaGSTfusionproteinwithamutatedinteractiondomain,4)TotestforbindingbetweentheputativeproteinandGST.MethodPreclearingthecelllysate---celllysate+glutathioneagarosebeads+GSTProbingthecelllysate---DetectinginteractingproteinsIncubation,40C,2hEnd-over-endmixingCentrifugation,40CsupernatantPreclearedcelllysate+glutathioneagarosebeads+GSTPreclearedcelllysate+glutathioneagarosebeads+GSTfusionprobeproteinIncubation,40C,2hEnd-over-endmixingCentrifugation,40CWashthebeads3timesElutetheGSTfusionprotein,anyproteinsoptionalMixthebeadsortheelutedproteinwithSDS-PAGEgelloadingbufferthesamplesboilingSDS-PAGEanalysisDetecting(Westernblotting,autoradiographyorproteinstaining).TooptimizetheGSTpull-downtechniqueforeachproteincomplex:Thebuffer(suchasRIPA)inwhichtheinteractionstakeplace;Theamountoftargetproteinmixedwiththefusion(probe)protein;Thecondictionsusedtowashthebeadstoeliminatenonspecificinteractions.TroubleshootingGSTpull-downexperimentTheGSTpulldownisarelatedtechniqueinwhicheither1)asingledefinedin-vitro-expressedprotein,2)anunknownproteinpresentinapoolofproteinsinacelllysate,or3)anunknownproteinexpressedfromapoolofin-vitro-translatedcDNAsiscollectedbyitsinteractionwithafusionproteincomposedofthetargetproteinlinkedtoaGSTmoiety.Thistechniquecanalsobeusedtoderivesemiquantitativeestimatesoftheaffinityofprotein-proteininteractions.