一个经典的蛋白芯片操作流程：ExperimentalProceduresforProteinMicroarrays--------------------------------------------------------------------------------ChemicallyDerivatizedGlassSlides.AldehydeslideswerepurchasedfromTeleChemInternational(Cupertino,CA).BSA-NHSslides,displayingactivatedaminoandcarboxylgroupsonthesurfaceofanimmobilizedlayerofbovineserumalbumin(BSA),werefabricatedasfollows.10.24gN,N"-disuccinimidylcarbonate(100mM)and6.96mlN,N-diisopropylethylamine(100mM)weredissolvedin400mlanhydrousN,N-dimethylformamide(DMF).30CMT-GAPslides(CorningIncorporated,Corning,NY),displayingaminogroupsontheirsurface,wereimmersedinthissolutionfor3hratroomtemperature.Theslideswererinsedtwicewith95%ethanolandthenimmersedin400mlofphosphatebufferedsaline(PBS),pH7.5containing1%BSA(w/v)for12hratroomtemperature.TheslideswererinsedtwicewithddH2O,twicewith95%ethanol,andcentrifugedat200gfor1mintoremoveexcesssolvent.Theslideswerethenimmersedin400mlDMFcontaining100mMN,N"-disuccinimidylcarbonateand100mMN,N-diisopropylethylaminefor3hratroomtemperature.Theslideswererinsedfourtimeswith95%ethanolandcentrifugedasabovetoyieldBSA-NHSslides.Theslideswerestoredinadesiccatorundervacuumatroomtemperatureforuptotwomonthswithoutnoticeablelossofactivity.--------------------------------------------------------------------------------ArrayingProteinsonGlassSlides.Proteinsweredissolvedin40%glycerol,60%PBS,pH7.5ataconcentrationof100μg/mlunlessindicatedotherwise.ForFigs.1,5,6,and7,theproteinswerespottedonaldehydeslidesusingaGMS417Arrayer(Affymetrix,SantaClara,CA).Followinga3hrincubationinahumidchamberatroomtemperature,theslideswereinvertedanddroppedontoasolutionofPBS,pH7.5containing1%BSA(w/v).After1min,theslideswereturnedrightsideupandimmersedintheBSAsolutionfor1hratroomtemperaturewithgentleagitation.FollowingabriefrinseinPBS,theslideswerereadyforfurtherprocessing(seebelow).ForFig.3,theproteinswerespottedonBSA-NHSslidesusingaGMS417Arrayer.Followinga3hrincubationinahumidchamberatroomtemperature,theslideswereinvertedanddroppedontoasolutionofPBS,pH8.0containing500mMglycine.After1min,theslideswereturnedrightsideupandimmersedintheglycinesolutionfor1hratroomtemperaturewithgentleagitation.Theslideswerethenreadyforfurtherprocessing(seebelow).ForFig.2,theproteinswerespottedonasinglealdehydeslideusingasplitpinarrayerconstructedfollowingdirectionsonP.Brown"swebpage(http://cmgm.stanford.edu/pbrown/).Followinga3hrincubationatroomtemperature,theslidewasprocessedusingtheprocedureemployedforthealdehydeslidesdescribedabove.--------------------------------------------------------------------------------ScreeningforProtein-ProteinInteractions.ProteinGwasfromPierce(Rockford,IL)andBODIPY-FL-Goat-anti-MouseIgGwasfromMolecularprobes(Eugene,OR).IKBalphaandp50werekindlyprovidedbyT.Maniatis(HarvardUniversity,Cambridge,MA)andGST-FRBand(His)6-FKBP12wereproducedrecombinantlyinEscherichiacoli.IKBalphaand(His)6-FKBP12werelabeledwithCy3andCy5,respectively,usingmonofunctionalreactivedyefromAmershamPharmaciaBiotech(Newark,NJ)andfollowingtherecommendedprotocol.ForFig.1,proteinG,p50,andFRBwerespottedinquadruplicateonaldehydeslidesandprocessedasdescribedabove.Toprobetheslides,thelabeledproteinsweredilutedintoPBS,pH7.5supplementedwith0.1%Tween-20(v/v)and1%BSA(w/v).BODIPY-FL-IgGwasusedataconcentrationof0.5μg/ml,Cy3-IKBalphawasusedataconcentrationof0.1μg/ml,andCy5-FKBP12wasusedataconcentrationof0.5μg/ml.0.55mlofproteinsolutionwasappliedtotheslideusingaPC500CoverWellincubationchamberfromGraceBiolabs(Bend,OR).Followinga1hrincubationatroomtemperature,theslideswererinsedwithPBSandthenwashed3timesfor3mineachwithPBST(PBSsupplementedwith0.1%Tween-20).TheslideswererinsedtwicewithPBSandcentrifugedat200gfor1mintoremoveexcessbuffer.Tovisualizefluorescence,theslideswerescannedusinganArrayWoRxfluorescenceslidescanner(AppliedPrecision,Issaquah,WA).Thescannerworksbyimagingsuccessive2.5x2.5mmsectionsofaslideusingexcitationandemissionfilterscoupledwithamagnifyinglensandCCDcamera.Theresultingpanelsarethenstitchedtogethertoformonelargeimage.Theslideswerevisualizedat5μmresolution,usingCCDcameraintegrationtimesrangingfrom1to5secdependingonthefluorophore.Theemittedlightwasfalse-coloredblue,green,andredtocorrespondtoBODIPY-FL,Cy3,andCy5,respectively.Forallimages,theintensityofthecolorwasscaledlinearly,withblackcorrespondingtothebackgroundfluorescenceoftheslideandpurecolorcorrespondingtothebrightestpixelsintheimage.ForFig.2,proteinGandFRBwerespottedonanaldehydeslide,probedwithBODIPY-FL-IgG+Cy5-FKBP12+100nMrapamycin,andvisualizedwithanArrayWoRxfluorescenceslidescanner,allasdescribedabove.ForFig.6,FRB(1mg/ml)wasspottedintriplicateon12separateareasoftwoaldehydeslides.Theareaswerethenseparatedbydrawinglinesbetweenthemwithahydrophobicpen(PAPPENfromNewcomerSupply,Middleton,WI).Theslideswereprocessedasdescribedabove.Toprobetheslides,Cy5-FKBP12wasseriallydiluted2-foldintoPBSTcontaining1%BSA(w/v)and1μMrapamycin.30μlofeachdilutionwereappliedtoseparatesectionsoftheslides.Followinga1hrincubationatroomtemperature,theslideswerewashedasdescribedaboveandscannedwithaGenePix4000Amicroarrayscanner(AxonInstruments,FosterCity,CA).Thefluorescenceintensityofeachspotwastakenasthemedianintensityofthespotminusthemedianintensityofthelocalbackground.InordertospanthefullrangeofintensitiesobservedatdifferentconcentrationsofCy5-FKBP12,thetwoslideswerescannedatdifferentsensitivitysettings(PMTvoltage)andthedatascaledtoadjustforthisdifference.--------------------------------------------------------------------------------ScreeningforSubstratesofProteinKinases.AllproteinsusedforthesestudieswerepurchasedfromNewEnglandBiolabs(Beverly,MA).EasyTidesgamma-33P-adenosine5"-triphosphate(gamma-33P-ATP)wasfromNENLifeScienceProducts(Boston,MA).NTB-2autorADIographyemulsion,Dektoldeveloper,andFixerwerefromEastmanKodakCompany(Rochester,NY).Kemptide,I-2,andElk1werespottedinquadruplicateonBSA-NHSslidesandprocessedasdescribedabove.Theslideswerethenwashed3timesfor10mineachwithWashBuffer(WB;20mMTris,150mMNaCl,10mMEDTA,1mMEGTA,0.1%TritonX-100,pH7.5).Theslidesweresubsequentlywashedoncefor10minwithKinaseBuffer(KB;50mMTris,10mMMgCl2,1mMDTT,pH7.5),incubatedfor10minwithKBsupplementedwith100μMATP,andwashedforanadditional10minwithKB.Theslideswerethenincubatedfor1hratroomtemperaturewith200μlofkinasesolution,appliedtotheslidesunderaPC200CoverWellincubationchamber(GraceBiolabs).ThekinasesolutionwascomposedoftherecommendedbufferforeachkinasesupplementedwiththerecommendedamountofATP,2μlofgamma-33P-ATP(20μCi),and2μlofpurifiedenzyme(10unitsofcAMP-dependentproteinkinase(catalyticsubunit),1000unitsofcaseinkinaseII,or100unitsofErk2).Followingthe1hrincubation,theslideswerewashed6timesfor5mineachwithWB,twicefor5mineachwithWBlackingTritonX-100,and3timesfor3mineachwithddH2O.Theslideswerethencentrifugedat200gfor1mintoremoveexcesswater.Tovisualizetheradioactivedecay,NTB-2autoradiographyemulsionwasmeltedat45oCfor45mininadarkroom.Theslidesweredippedintheemulsionfor3secandallowedtodryverticallyatroomtemperaturefor4hr.Theslideswerethensealedina?-radiationboxwithdesiccantandincubatedinthedarkat4oCfor4to10days.TheslidesweresubsequentlydevelopedbyimmersingthemsuccessivelyinDektoldeveloperfor2min,ddH2Ofor10sec,Fixerfor5min,andddH2Ofor5min.Tovisualizetheslides,successiveimagesweretakenusingaDeltaVisionautomatedmicroscope(AppliedPrecision)inDICmodeandtheindividualpanelsstitchedtogethertoformasinglelargerimage.Thesamesettingswereusedforallthreeslides.--------------------------------------------------------------------------------ScreeningforTargetsofSmallMolecules.Mouseanti-digoxigeninIgGclone1.71.256(Anti-DIG)wasfromBoehringerMannheim(Indianapolis,IN),streptavidinwasfromPierce,andbovineserumalbumin(BSA)wasfromSigma(St.Louis,MO).Alexa488-BSAwaspreparedusinganAlexaFluor488proteinlabelingkitfromMolecularProbes.Alexa488-BSA-DigwaspreparedbylabelingAlexa488-BSAwith3-amino-3-deoxydigoxigeninhemisuccinamide,succinimidylester(MolecularProbes).Cy3-BSAandCy5-BSAwerepreparedbylabelingBSAwithCy3andCy5monofunctionalreactivedyes(AmershamPharmaciaBiotech).Cy5-BSA-biotinwaspreparedbylabelingCy5-BSAwithSulfo-NHS-LC-Biotin(Pierce).Cy3-BSA-maleimidewaspreparedbylabelingCy3-BSAwithSulfo-GMBS(Pierce).Alllabelingreactionswereperformedaccordingtotherecommendedprotocols.Cy3-BSA-AP1497,Cy3-BSA-AP1767,andCy3-BSA-AP1780werepreparedasfollows.AP1497,AP1767,andAP1780werekindlyprovidedbyD.Holt(AriadPharmaceuticals,Cambridge,MA).Eachcompoundwascoupledtopolystyrenebeadsviaa6-carbonlinkerand4-methoxytrityl-protectedcysteineaccordingtoourpreviouslypublishedprotocol(http://www.cgr.harvard.edu/macbeath/protocols/smmicroarrays.html).Foreachcompound,about15beadswereincubatedin100μlofa17:2:1mixtureofchloroform,trifluoroaceticacid,andtriethylsilanefor2hratroomtemperature.Thecleavagesolutionwasthenremovedinvacuo,leavingabout750nmolofeachcompound.About165equivalentsofthiol-labeledsmallmoleculewasincubatedwithCy3-BSA-maleimideinPBSfor6hratroomtemperature.Followinga1hrincubationwith200mM2-mercaptoethanol,theconjugatesweredialyzedextensivelyagainstPBS,yieldingCy3-BSA-AP1497,Cy3-BSA-AP1767,andCy3-BSA-AP1780.Anti-DIG,streptavidin,andFKBP12werespottedinquadruplicateonaldehydeslidesandprocessedasdescribedabove.Toprobetheslides,thedoublylabeledBSAconjugatesweredilutedintoPBSTsupplementedwith1%BSA(w/v)ataconcentrationof10μg/ml.0.55mlofproteinsolutionwasappliedtotheslide,usingaPC500CoverWellincubationchamber.Followinga1hrincubationatroomtemperature,theslideswererinsedwithPBSandthenwashed3timesfor3mineachwithPBST.TheslideswererinsedtwicewithPBS,centrifugedat200gfor1mintoremoveexcessbuffer,andimagedonanArrayWoRxfluorescenceslidescannerasabove.

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