HowtoplananimmunoprecipitationofyourGFP-fusionproteinwhenusingtheChromoTekGFP-Trap®

PreambleThisdocumentprovidespracticalinformationonhowtoapplytheChromoTekGFP-Trap®forimmunoprecipitation.

IntroductionTheChromoTekGFP-Trap®isbasedonaGFP-bindingproteinderivedfromanAlpacasinglevariabledomainantibody,alsocalledVHHornanobody(figure1).TheGFP-TraphasparticularpropertiesandprovidessomeadvantagesovertrADItionalIgGantibodieswhenappliedinimmunoprecipitations.

PlanningoftheexperimentTherearesomeexperimentalaspectsthatyoushouldconsiderwhenplanningimmunoprecipitationofyourGFP-fusionproteinofinterestusingtheChromoTekGFP-Trap®.Below,weguideyouthrougheachstep.

Thereare3matricesavailable:

Pre-clearing:Pre-clearingisanoptionalsteptoremoveproteinsorDNAwhichbindnon-specificallytothesolid-phasesupport.Itincludestheincubationofthecellextractwithplainbeads(e.g.bindingcontrolagarosebeads)beforeperformingtheactualimmunoprecipitationexperimentwiththeGFP-Trap.Aftersuccessfulpre-clearing,non-specificproteinsorothercomponentswillnotbeco-purifiedwiththeproteinofinterest. CRAPome:Bynature,everymatrixandbindingmoleculemaynonspecificallybindsomeproteinsresultinginproteinbackground.Scientistshaveestablishedtheinternet-baseddatabaseCRAPomeatwww.crapome.org.Thisdatabasestoresandannotatesnegativecontrolsgeneratedbytheproteomicsresearchcommunity.CRAPomehelpstodeterminethebackgroundcontaminants--forexample,proteinsthatinteractwiththesolid-phasesupport,affinityreagentorepitopetag. Specificity-WhatfluorescentproteinsarecapturedbytheGFP-Trap®TheGFP-Trap®specificallybindstomostofthecommonGFPderivatives:

TheChromoTekGFP-Trap®onlybindsproperlyfoldedactiveGFP.Itisbelievedthatthisisbecausethatnanobodybindstoathree-dimensionalepitopeofGFP.Thenanobody’selongatedCDR3(complementaritydeterminingregion3)allowstoreachintocleftsandenzymaticcentersofproteins,whicharenotaccessIBLetoconventionalantibodiesbutresultsinverystrongbindingandverylowdissociationconstantsofthisGFPnanobody.Thereforethatanti-GFPnanobodyisnotsuitableforproteindetectioninWesternBlots.ForWesternBlotdetectionofGFP(fusionproteins)ChromoTekrecommendsthetraditionalantibodyanti-GFPantibody3H9(ratmonoclonal,seecomplementaryproducts). Controls–WhatcontrolsshouldIconducttovalidatetheexperimentaldata?Belowfindsomesuggestionsbyapplication:ForImmunoprecipitation(IP):•GFP-Trap®forIPofGFP-fusionsandanon-relevantNano-Trapasnegativecontrol,e.g.Myc-Trap®,GST-TraporMBP-TrapForCo-Immunoprecipitation(Co-IP)ofproteincomplexAB: CellLysis–Whattoconsiderwhenpreparingacelllysate?Lysisbuffers:•Anon-denaturinglysisbufferissuitableforCo-IP,becauseproteinswillremainintheirnativeconformation•TheRIPA(RadioImmunoprecipitationAssay)buffermightdenatureproteinsordisruptproteincomplexesInhibitors:•Addproteaseinhibitorstopreventproteolysis!•Preserveposttranslationalmodificationsofyourproteinandadde.g.phosphataseinhibitors!•Preventdegradationofyourproteinbykeepingyoursamplesonice! Immunoprecipitation–BindingoftheGFP-fusionSincetheGFPbindingproteiniscovalentlycoupledtothebeads’surface,theGFP-Trap®beadsareready-to-useandcanbedirectlyaddedtothepreparedlysate.TheaffinityofGFP-nanobodyisinthepicomolarrange,thereforedepletionofGFP-fusionscanbecompletedwithin5-30minutes. BuffercompatibilityoftheGFP-Trap®forbindingandwashingTheGFP-Trap®iscompatiblewithmostwashbuffersandstableunderharshconditionsincluding:•Upto1MNaCland8MUrea•Upto0.2%SDSand2%NP-40 ElutionstrategiesTheelutionoftheboundGFP-fusionproteinbyacompetitivepeptide,whichreplacestheGFP-fusionproteindoesn’twork.Also,theadditionofchaotropiccompoundslikeureadon’telutethebondGFP-fusionproteinastheGFP-Trap®worksunderdenaturingconditions.Wethereforerecommendtoelutewith: •SDS,e.g.SDSsamplebuffer,isaveryeffectivewaytoelutetheboundGFP-taggedprotein.TheelutionresultsindenaturedGFP-fusions.•0.2MglycinepH2.5 AlternativelyyoumayelutewithglycineatpH2.5.ItisrecommendedtorepeatthiselutionstepasthepHshiftelutionworksincompletely.Therepetitionwillimprovetheelutionefficiency. Veryimportant:Don’tforgettoneutralizeproteinsimmediatelyafterelution! Asanalternativetoaboveelutionoptions,aproteasecleavagesitebetweenGFPandthefusionproteincanbeintroduced.Thisoptionisrecommendediflessstableproteinshavebeenboundorifyouwanttoenrichyournativeproteinofinterest.FurThermore,considerwhetheryoureallyneedtoelutetheboundproteinofinterestfromthebeadsratherthanconductthedownstreamanalysis\"on-bead”:•Proteinscanbedigestedwhenstillcoupledtothebeadsforsubsequentmassspectrometryanalysis.•Enzymaticactivityassayscanbeperformedwhenstillcoupledtothebeadsiftheactivecenterisnotblocked. ReproducibilityTheGFP-Trap®isasmall,solubleandstablesinglepolypeptidechainthatisrecombinantlyexpressedinbacteria.Thisincombinationwithqualitycontrolmakesitsproductionrobustandreproducibleforreliableresults. SelectedReferencestointroduceNanobodiesandtheirapplications:NanobodiesasprobesforproteindynamicsinvitroandincellsDmitriev,O.Y.,Lutsenko,S.andMuyldermans,S.in:JournalofBIOLOGicalChemistry,2015–jbc-R115. AversatilenanotrapforbiochemicalandfunctionalstudieswithfluorescentfusionproteinsRothbauer,U.,Zolghadr,K.,Muyldermans,S.,Schepers,A.,Cardoso,M.C.,Leonhardt,H.in:MolCellProteomics,2008Feb;7(2):282-9.Epub2007Oct21 Nanobody-basedproductsasresearchanddiagnostictoolsDeMeyer,T.,Muyldermans,S.andDepicker,A.in:TrendsinBiotechnology,2014May;32(5):263-270; Beneficialpropertiesofsingle-domainantibodyfragmentsforapplicationinimmunoaffinitypurificationandimmuno-perfusionchromatographyVerheesenP.,TenHaaftM.R.,LindnerN.,VerripsC.T.,deHaardJ.J.W.in:Biochim.Biophys.Acta,2003;1624(1–3):21–28 Ahighlyspecificgoldnanoprobeforlive-cellsingle-moleculeimagingLeduc,C.,Si,S.,Gautier,J.,Soto-Ribeiro,M.,Wehrle-Haller,B.,Gautreau,A.,.Giannone,G.,Cognet,L.&Lounis,B.in:Nanoletters,2013:13(4),1489-1494. Nanobody-basedchromatinimmunoprecipitation.Duc,T.N.,Hassanzadeh-Ghassabeh,G.,Saerens,D.,Peeters,E.,Charlier,D.,&Muyldermans,S.in:SingleDomainAntibodies:MethodsandProtocols,2013:491-505