HowtoplananimmunoprecipitationofyourGFP-fusion
proteinwhenusingthechromotekGFP-Trap®

 

Preamble
ThisdocumentprovidespracticalinformationonhowtoapplytheChromoTekGFP-Trap®forimmunoprecipitation.

 

Introduction
TheChromoTekGFP-Trap®isbasedonaGFP-bindingproteinderivedfromanAlpaca
singlevariabledomainantibody,alsocalledVHHornanobody(figure1).TheGFP-TraphasparticularpropertiesandprovidessomeadvantagesovertrADItionalIgGantibodieswhenappliedinimmunoprecipitations.

Planningoftheexperiment
TherearesomeexperimentalaspectsthatyoushouldconsiderwhenplanningimmunoprecipitationofyourGFP-fusionproteinofinterestusingtheChromoTekGFP-Trap®.Below,weguideyouthrougheachstep.

 

Thereare3matricesavailable:

 

Pre-clearing:
Pre-clearingisanoptionalsteptoremoveproteinsorDNAwhichbindnon-specificallytothesolid-phasesupport.Itincludestheincubationofthecellextractwithplainbeads(e.g.bindingcontrolagarosebeads)beforeperformingtheactualimmunoprecipitationexperimentwiththeGFP-Trap.Aftersuccessfulpre-clearing,non-specificproteinsorothercomponentswillnotbeco-purifiedwiththeproteinofinterest.
 
CRAPome:
Bynature,everymatrixandbindingmoleculemaynonspecificallybindsomeproteins
resultinginproteinbackground.Scientistshaveestablishedtheinternet-baseddatabase
CRAPomeatwww.crapome.org.Thisdatabasestoresandannotatesnegativecontrols
generatedbytheproteomicsresearchcommunity.CRAPomehelpstodeterminethe
backgroundcontaminants--forexample,proteinsthatinteractwiththesolid-phasesupport,affinityreagentorepitopetag.
 
Specificity-WhatfluorescentproteinsarecapturedbytheGFP-Trap®
TheGFP-Trap®specificallybindstomostofthecommonGFPderivatives:

FeatureAgarosebeadsMagneticagarosebeadsMagneticbeads
MatrixAgarose,4%highly
cross-linked
Agarose,6%cross-linkedSilica,non-porous
Particalsize45-165μm20-40μm0.5-1μm
Bindingcapacity2-3μgper10μl
beadslurry
2-3μgper10μlbeadslurry0.5μgper10μlbead
slurry
Magneticnoyesyes

 

TheChromoTekGFP-Trap®onlybindsproperlyfoldedactiveGFP.Itisbelievedthatthisisbecausethatnanobodybindstoathree-dimensionalepitopeofGFP.Thenanobody’s
elongatedCDR3(complementaritydeterminingregion3)allowstoreachintocleftsand
enzymaticcentersofproteins,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-Trap
ForCo-Immunoprecipitation(Co-IP)ofproteincomplexAB:
 
CellLysis–Whattoconsiderwhenpreparingacelllysate?
Lysisbuffers:
    •Anon-denaturinglysisbufferissuitableforCo-IP,becauseproteinswillremainin
theirnativeconformation
    •TheRIPA(RadioImmunoprecipitationAssay)buffermightdenatureproteinsor
disruptproteincomplexes
Inhibitors:
    •Addproteaseinhibitorstopreventproteolysis!
    •Preserveposttranslationalmodificationsofyourproteinandadde.g.phosphatase
inhibitors!
    •Preventdegradationofyourproteinbykeepingyoursamplesonice!
 
Immunoprecipitation–BindingoftheGFP-fusion
SincetheGFPbindingproteiniscovalentlycoupledtothebeads’surface,theGFP-Trap®beadsareready-to-useandcanbedirectlyaddedtothepreparedlysate.TheaffinityofGFP-nanobodyisinthepicomolarrange,thereforedepletionofGFP-fusionscanbecompletedwithin5-30minutes.
 
BuffercompatibilityoftheGFP-Trap®forbindingandwashing
TheGFP-Trap®iscompatiblewithmostwashbuffersandstableunderharshconditions
including:
    •Upto1MNaCland8MUrea
    •Upto0.2%SDSand2%NP-40
 
Elutionstrategies
TheelutionoftheboundGFP-fusionproteinbyacompetitivepeptide,whichreplacesthe
GFP-fusionproteindoesn’twork.Also,theadditionofchaotropiccompoundslikeureadon’telutethebondGFP-fusionproteinastheGFP-Trap®worksunderdenaturingconditions.Wethereforerecommendtoelutewith:
 
    •SDS,e.g.SDSsamplebuffer,isaveryeffectivewaytoelutetheboundGFP-tagged
protein.TheelutionresultsindenaturedGFP-fusions.
    •0.2MglycinepH2.5
      AlternativelyyoumayelutewithglycineatpH2.5.Itisrecommendedtorepeatthis
elutionstepasthepHshiftelutionworksincompletely.Therepetitionwillimprovethe
elutionefficiency.
      Veryimportant:Don’tforgettoneutralizeproteinsimmediatelyafterelution!
 
Asanalternativetoaboveelutionoptions,aproteasecleavagesitebetweenGFPandthefusionproteincanbeintroduced.Thisoptionisrecommendediflessstableproteinshave
beenboundorifyouwanttoenrichyournativeproteinofinterest.
FurThermore,considerwhetheryoureallyneedtoelutetheboundproteinofinterestfromthebeadsratherthanconductthedownstreamanalysis“on-bead”:

    •Proteinscanbedigestedwhenstillcoupledtothebeadsforsubsequentmass
spectrometryanalysis.
    •Enzymaticactivityassayscanbeperformedwhenstillcoupledtothebeadsifthe
activecenterisnotblocked.
 
Reproducibility
TheGFP-Trap®isasmall,solubleandstablesinglepolypeptidechainthatisrecombinantlyexpressedinbacteria.Thisincombinationwithqualitycontrolmakesitsproductionrobustandreproducibleforreliableresults.
 
 
SelectedReferencestointroduceNanobodiesandtheirapplications:
Nanobodiesasprobesforproteindynamicsinvitroandincells
Dmitriev,O.Y.,Lutsenko,S.andMuyldermans,S.in:JournalofBIOLOGicalChemistry,2015–jbc-R115.
 
Aversatilenanotrapforbiochemicalandfunctionalstudieswithfluorescentfusionproteins
Rothbauer,U.,Zolghadr,K.,Muyldermans,S.,Schepers,A.,Cardoso,M.C.,Leonhardt,H.in:MolCellProteomics,2008Feb;7(2):282-9.Epub2007Oct21
 
Nanobody-basedproductsasresearchanddiagnostictools
DeMeyer,T.,Muyldermans,S.andDepicker,A.in:TrendsinBiotechnology,2014May;32(5):263-270;
 
Beneficialpropertiesofsingle-domainantibodyfragmentsforapplicationinimmunoaffinitypurificationandimmuno-perfusionchromatography
VerheesenP.,TenHaaftM.R.,LindnerN.,VerripsC.T.,deHaardJ.J.W.in:Biochim.Biophys.Acta,2003;1624(1–3):21–28
 
Ahighlyspecificgoldnanoprobeforlive-cellsingle-moleculeimaging
Leduc,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

FluorescentproteinGFP-Trap® FluorescentproteinGFP-Trap®
(e)GFP(e)Citrine
tagGFP2AcGFP
turboGFP-SuperfolderGFP
mClovermCerulean
YFPpHluorin
CFPGFPS65T
Venus--



   ProductnameSizeCode
     GFP-Trap®A
      ▶coupledtoagarosebeads
10rxns(250µlresin)gta-10
20rxns(500µlresin)gta-20
100rxns(2.5mlresin)gta-100
200rxns(5mlresin)gta-200
400rxns(10mlresin)gta-400
     GFP-Trap®AKit
      ▶GFP-Trap®A
      ▶incl.lysiswashandelutionbuffers
20rxns(500µlresin)gtak-20
     GFP-Trap®MA
       ▶coupledtomagneticagarosebeads
10rxns(250µlresin)gtma-10
20rxns(500µlresin)gtma-20
100rxns(2.5mlresin)gtma-100
200rxns(5mlresin)gtma-200
400rxns(10mlresin)gtma-400
     GFP-Trap®MAKit
      ▶GFP-Trap®MA
      ▶incl.lysis,washandelutionbuffers
20rxns(500µlresin)gtmak-20
     GFP-Trap®M
     ▶coupledtomagneticparticles
10rxns(250µlresin)gtm-10
20rxns(500µlresin)gtm-20
100rxns(2.5mlresin)gtm-100
200rxns(5mlresin)gtm-200
400rxns(10mlresin)gtm-400
     GFP-Trap®MKit
       ▶GFP-Trap®M
       ▶incl.lysis,washandelutionbuffers
20rxns(500µlresin)gtmk-20
     GFP-multiTrap®
       ▶black96wellplate
96rxns(1plate)gtp-96
480rxns(5plate)gtp-480
     GFP-Trap®
       ▶uncoupledprotein
250µlgt-250
     Spincolumns10unitssct-10
20unitssct-20
50unitssct-50
     Bindingcontrol
       ▶agarosebeads
500µl(20rxns)bab-20
     Bindingcontrol
       ▶magneticagarosebeads
500µl(20rxns)BMAb-20
      Bindingcontrol
       ▶magneticparticles
500µl(20rxns)bmp-20
     GFPantibody(3H9)
       ▶ratmonocional
100µl3H9