·YeastTwo-HybridSystem(FinleyLab)Thisisoneofthemostcomprehensiveanddetailedguidetoyeasttwo-hybridsystemtechniquewithintroductiontoitsbackground.Thefollowingproceduresaredescribed:matingassay-smallscalefortensofdifferentbaitorpreyproteins,collectingbait(andprey)strains,largescaleforhundredsofdifferentbaitorpreystrains,interactionmatingassaywithotheryeasttwo-hybridsystems,recordingresults,interpretinginteractiondata,qualitativeinterpretationandmore...

·YeastTwo-HybridSystem(FinleyLab)Theupdatedversionforscalingupthethesetechniquestofacilitatecharacterizationoflargenetworksofinteractingproteins.Itisalsousefulforindividualhunts.

·PerformingaHuntbyInteractionMating(FinleyLab)InthisprotocolonestrainistransformedwithlibraryDNAandthetransformantsarecollectedandfrozeninaliquots.Foreachinteractorhunt,analiquotofthisfrozen"pretransformedlibrarystrain"isthawedandmixedwithanaliquotofabaitstraintransformedwiththebaitexpressionplasmid.OvernightincubationofthemixtureonaYPDplateresultsinmating-i.e.,cellsofonestrainfusewithcellsoftheotherstraintoformdiploids.Thediploidsarethenexposedtogalactosetoinduceexpressionofthelibraryencodedproteins,andinteractorsareselectedinthesamemannerasinthestandardhuntprotocols.

·GSTFusionProteinPurificationfromYeast(DohlmanLab)

·TwoHybridSystemTransformation(GietzLab)ThisProtocolisfor2Hybridsystemscreens

·PurificationofMBP(maLTose-BindingProteins)FusedProteins(PMCI)

• PurificationofHis-taggedproteinsfromSf9cellsundernativeconditions(BrentGraveley)

·PurificationofGSTFusedProteins(PMCI)