《pulldown技术》PPT课件

DetectionofProtein-ProteinInteractionsUsingtheGSTFusionProteinPull-downTechnique,BacteriallyexpressedglutathioneS-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.,GST,ProteinX,GST,35S-labledcelllysate,(glutathione-sepharosebeads),(glutathione-sepharosebeads),35S-labledcelllysate,GST,ProteinX,GST,GST,Interactat40C,Microfugetocollectcomplexes,123,autoradiograph,AnalyzebySDS-PAGE,Lane1.MarkerLane2.GST-proteinXLane3.GST,TheschemaofaGSTpull-downexperiment,GST,GST,ProteinX,Twogeneraluses:Toidentifynovelinteractionsbetweenafusion(orprobe)proteinandunknown(ortarget)proteins;Theproteinconcentrations,Istheprobeproteinnormallyexpressedinthatparticularcellortissue?Isthegoaltocomparedifferenttypesofcellpopulations?Toconfirmsuspectedinteractionsbetweentheprobeproteinandaknownproteins.antibodiestothetargetprotein,or:the35S-labeledinvitrotranslatedprotein,orthetargetproteincanbetaggedwithanepitope;Cellinculturecanbetransfectedwithaplasmidencodingthetargetproteintoincreasetheabundance;Tocontrolthespecificityofbinding,thebestistheinclusionofaGSTfusionproteinwithamutatedinteractiondomain,TotestforbindingbetweentheputativeproteinandGST.,Method,Preclearingthecelllysate-celllysate+glutathioneagarosebeads+GSTProbingthecelllysate-Detectinginteractingproteins,Incubation,40C,2h,End-over-endmixing,Centrifugation,40C,supernatant,Preclearedcelllysate+glutathioneagarosebeads+GST,Preclearedcelllysate+glutathioneagarosebeads+GSTfusionprobeprotein,Incubation,40C,2h,End-over-endmixing,Centrifugation,40C,Washthebeads3times,ElutetheGSTfusionprotein,anyproteins,optional,MixthebeadsortheelutedproteinwithSDS-PAGEgelloadingbuffer,thesamples,boiling,SDS-PAGEanalysis,Detecting(Westernblotting,autoradiographyorproteinstaining).,TooptimizetheGSTpull-downtechniqueforeachproteincomplex:Thebuffer(suchasRIPA)inwhichtheinteractionstakeplace;Theamountoftargetproteinmixedwiththefusion(probe)protein;Thecondictionsusedtowashthebeadstoeliminatenonspecificinteractions.TroubleshootingGSTpull-downexperiment,TheGSTpulldownisarelatedtechniqueinwhicheither1)asingledefinedin-vitro-expressedprotein,2)anunknownproteinpresentinapoolofproteinsinacelllysate,or3)anunknownproteinexpressedfromapoolofin-vitro-translatedcDNAsiscollectedbyitsinteractionwithafus