| cAMPAssays |
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| GouzelKarimovaandDanielLadantUnitePostulantedeBiochimiedesInteractionsMacromoleculaires,DepartementdeBIOLOGieStructuraleetChimie,CNRSURA2185,InstitutPasteur,Paris,FranceExcerptedfromProtein:ProteinInteractions,SecondEditionEditedbyEricaA.GolemisandPeterD.Adams |
| ABSTRACT |
| cAMPmeasurementsareobtainedusinganELISAassay(HarlowandLane1988).CommercialrADIo-immunoassays,orELISAkits,toassaycAMPcanbepurchasedfromvariousmanufacturers.Inourlaboratoryweusethehomemade,less-expensiveELISAthatisdescribedbelow.Inoutline,thisassayisbasedontheABIlityofsolublecAMPinbacterialextractstocompetewithbindingofanalkalinephosphatase(AP)-conjugatedanti-cAMPantibodytoimmobilizedcAMP.Thesurface-bound,immobilizedanti-cAMP-APisinverselycorrelatedtotheconcentrationofcAMPintheextract.KnownconcentrationsofsolublecAMPareusedtocalibratetheassay. |
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| MATERIALS |
| Buffers,Solutions,andReagents |
- APsubstrate,5´-para-nitrophenylphosphate(PNP)
- BSA,1mg/µlin200mMHEPES-Na(pH7.5)
- BSA,20mg/mlinHBSTbuffer
- Bovineserumalbumin(BSA)at1mg/mlin200mMHEPES-NaOH(pH7.5)
- Coatingbuffer:0.1MNa2CO3(pH9.5)
- Dimethylformamide(DMF)
- N-Ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline(EEDQ)
- HBSTbuffer
- 50mMHEPES(pH7.5)
- 150mMNaCl
- 0.1%Tween-20
HEPES-NaOH,10mM(pH7.5)in100mMNaClHEPES-NaOH,100mM(pH7.5)O2´Monosuccinyladenosine3´:5´-cyclicmonophosphate(O2´-Suc-cAMP) |
| Cells |
- BacterialsstrainstobeassayedforcAMP(exponentiallygrowingorovernightculture)
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| Antibodies |
- Rabbitanti-cAMPantiserum
- Goatanti-rabbitIgGcoupledtoalkalinephosphatase(AP)
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| SpecialEquipment |
- ELISAplate
- Boilingwaterbathorheatingblock,presetto100°C
- Incubatorpresetto30°C
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| AdditionalEquipmentandReagents |
- ThisprocedurealsorequiresequipmentfordialysisandforthepreparationofcAMP-BSAconjugate(seestep1fordetails).
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| METHOD |
- PreparecAMP-BSAconjugateasfollows(modifiedfromJosephandGuesdon1982).
- PrepareasolutionofEEDQat100mg/mlinDMF(e.g.,10mgofEEDQinto100µl).
- Preparea60mMsolutionofO2´-Suc-cAMPin100mMHEPES-NaOH(pH7.5).
- Incubate500µloftheO2´-Suc-cAMPsolutionwith75µloftheEEDQsolutionfor45minatroomtemperature.Thenadd500µlofasolutionofBSAat1mg/mlin200mMHEPES-Na(pH7.5)andincubateovernightatroomtemperature.
- Dialyzethemixtureextensivelyagainst10mMHEPES-NaOH(pH7.5),100mMNaCl.
- StorethecAMP-BSAconjugateat-20°C(forseveralyears),orat4°C(forseveralweeks).AsimilarcAMPconjugatewasusedasanimmunogentoraiseanti-cAMPantibodiesinrabbit(JosephandGuesdon1982).
DilutethecAMPconjugate5,000-10,000times(optimaldilutionshouldbedeterminedexperimentally)incoatingbufferandadd50lofthedilutiontoeachwellofanELISAplate.Incubateovernightat4°C.ThiswillcoattheELISAplatewithcAMPconjugate.Thenextday,washthewellsoftheELISAplatewithHBSTbufferandadd250µlofBSA(20mg/mlinHBSTbuffer)toeachwell.Incubatefor1hratroomtemperatureandthenwashthewellswithHBSTbuffer.Thiswillblocknonspecificprotein-bindingsites.Preparethecellextractsbyboiling1ml(in1.5-mlmicrotubes)ofbacterialculturesfor5min.Thencentrifugethetubesat13,000gfor10mintoremovethecelldebris.Add100µl/welloftheboiledcellextracts(eitherundilutedorafterserialdilutionswithHBSTbuffer)tothecAMP-BSA-coatedELISAplate.Inparallel,add100µlofknownconcentrationcAMPsolution(concentrationrangingfrom0to3000nM,e.g.,0,3,10,30,100,300,1000,3000nMinLBmedium)toeachwellofthecAMP-BSA-coatedELISAplate.Diluterabbitanti-cAMPantiserum1,000-10,000times(optimaldilutionshouldbedeterminedexperimentally)inHBSTbuffercontaining10mg/mlBSA.Add50µlofthedilutedantiserumtoeachwell.Incubatetheplateovernightat4°C(orfor2-3hrat30°C).Washtheplateextensively(3x5min)with300µl/wellofHBST.Dilutegoatanti-rabbitIgGcoupledtoAP5,000-10,000times(optimaldilutionshouldbedeterminedexperimentally)inHBSTbufferandadd100µltoeachwelloftheELISAplate.Incubatefor1hrat30°C.Washtheplateextensively(3x5min)with300µlperwellofHBSTTorevealAPactivity,add100µlofPNPsubstratetoeachwellandincubatefor20-200minat30°Cuntilsufficientcolordevelops.MeasuretheODineachwellat405nmonamicrotiterplatereader.ForallsamplescorrespondingtotheknowncAMPconcentrations,plottheOD405asafunctionofcAMPconcentrationtoobtainastandardcurve.Ifcommercialdataanalysis/graphpresentationsoftwareisavailable(e.g.,MicrosoftExcel,KaleidaGraph,SigmaPlot),itisconvenienttofitthedatatoasigmoidcurveusingtheequationOD405=ODMx(1-Cn/[Cn+K])whereOD405istheODmeasuredforthesamplescontainingthecAMPconcentrationC,andODMtheODmeasuredforthesampleswithoutcAMP,Ktheequilibriumconstant,andnacooperativityfactor.Theselasttwonumbers,Kandn,aredefinedasvariablesthatwillbefittedbythesoftware.Notethatthecooperativityfactor,n,isusuallycloseto1andcaneventuallybeomittedfromtheequation.ThisequationcanthenbeusedtodeterminethecAMPconcentrationforalltestedsamplesfromtheOD405measurement.Alternatively,approximatecAMPconcentrationsintheunknownsamplescanbededucedusingaplottedstandardcurveandthemeasuredOD405readings. |
| REFERENCES |
| HarlowE.andLaneD.1988.Antibodies:ALaboratoryManual.ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p.579.JosephE.andGuesdonJ.L.1982.Beta-galactosidaseimmunoassayforthemeasurementofcyclicAMP.Anal.Biochem.119:335-340. |
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| Anyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliabilityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,pleaseconsult:Protein:ProteinInteractions,SecondEdition:AMolecularCloningManual,editedbyEricaA.GolemisandPeterD.Adams,©2005byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p.513. |
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| Copyright©2006byColdSpringHarborLaboratoryPress.Allrightsreserved.Nopartofthesepages,eithertextorimagemaybeusedforanypurposeotherthanpersonaluse.Therefore,reproductionmodification,storageinaretrievalsystemorretransmission,inanyformorbyanymeans,electronic,mechanical,orotherwise,forreasonsotherthanpersonaluse,isstrictlyprohibitedwithoutpriorwrittenpermission. |
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