Mediomics/Bridge-It® cAMP-Phosphodiesterase (PDE) Assay Kit, 384-well format/PD1007

Bridge-It®Phosphodiesterase(PDE)AssayKit,384-wellformat

TheBridge-It®Phosphodiesterase(PDE)Assayisafluorescence-based,high-throughputscreening(HTS)methodformeasuringcyclicnucleotidephosphodiesteraseactivityfromapurifiedsourceanddeterminingtheIC50oftheirinhibitors.ThisassayisbasedontheformatofBridge-It®cAMPallinoneFluorescenceAssay(Mediomics,Cat#122938).

Applications:

• PDE-relateddrugdiscoveryprojects

• Researchon heart,lungs(i.e.COPD),plateletfunction,andinflammatorymechanisms

• Researchonanxietydisordersorneurodegenerativediseases

Features:

• Easy: MixtestsampleorstandardwiththeAssaysolutionandincubateat~25°C

• Fast: Readfluorescentsignalafter30minutesofincubation

• FlexIBLe: Assayisadaptabletobothlow-andhigh-throughputscreeningformats

Pricing: The100-wellkitisaone-timetrialpurchase.Forbulkpricingoptions,please contactus.Foranyotherquestionsorcomments,pleasedonothesitateto contactus.Wearealwayshappytohelp!

 BackgroundInformationoncAMP,PDE,andtheBridge-ItFluorescenceAssay

Adenosine-3’,5’-cyclicmonophosphate(cAMP)

Adenosine-3’,5’-cyclicmonophosphate(cAMP)isanimportantsecondmessengerwhichisinvolvedinthemodulationofnumerousBIOLOGicalprocesses.ThemeasurementofcAMPisespeciallyimportantinnewdrugdiscoverysincecAMPlevelsarecloselyrelatedtotheactivityofoneofthemajortargetsfornewdrugdiscovery–theGprotein-coupledreceptors(GPCR).MediomicsfluorescencecAMPassaymethodfordeterminingtheconcentrationsofcAMPisbasedontheassayplatformdesigndescribedabove.BasicallyCAP,abacterialDNAbindingproteinwhoseDNAbindingactivitydependsonthepresenceofcAMP,isusedasahighlyspecificbiosensorformeasuringcAMPlevels.AsPDEhydrolyzescAMPtoAMP,theconcentrationofcAMPdecreases,whichresultsinlessoftheCAP-DNAcomplexformationandmorefree“half-site”duplexeswithprobes(unquenched).TheincreaseinthefluorescencesignalisproportionaltothePDEactivity.

Bridge-It®PDEAssay–cAMPStandardCurves

ThefollowingcAMPstandardcurveisrepresentativeofthosepreparedusingtheBridge-It®PDEassay.ItshowsastandardcurvepreparedinBufferBina384-wellplatewiththefluorescencesignalreadafter30minutes,1hour,and2hoursafteradditionof2XAssaySolution.

Bridge-It®PDEAssay–PDEActivityAssay

ThefollowingPDEactivityassayisrepresentativeofthosepreparedusingtheBridge-It®PDEassay.The relativeactivitywasmeasuredinsampleswiththeindicatedamountofPDEfrombovinebrain(Sigma,St.Louis,MO,Cat#P9529).Thereactionwasincubatedatroomtemperaturefor30minutesandstoppedbyadditionof0.5ulstopsolution.The2Xassaysolutionwasaddedandthesignalwasreadafterincubationatroomtemperaturefor40minutes.

Bridge-It®PDEAssay–IC50Determination

ThefollowingassayisrepresentativeofthosepreparedusingtheBridge-It®PDEassay.The relativeactivitywasmeasuredinsampleswith0.8mUPDE frombovinebrain(Sigma,St.Louis,MO,Cat#P9529) andtheindicatedamountofIBMX (Sigma,St.Louis,MO).

Note:DefinitionofRA(relativeactivity)isonpage8oftheprotocol.ThePDE assaywasperformedinastandard384-wellplateinafinal10ulreactionin1xPDEreactionbuffer(25mMTris,pH7.4,100mMNaCl,BSA0.1mg/ml,CaCl20.03mM,Calmodulin(Sigma,St.Louis,MO,Cat#P1431)10U/ml),with0.8mUPDEfrombovinebrain(Sigma,St.Louis,MO,Cat#P9529)andtheindicatedamountofIBMX(Sigma,St.Louis,MO).TheenzymeandIBMXweremixedandpre-incubatedatroomtemperaturefor5minutes.6pmolecAMPwasadded,andthereactionswereincubatedfor30minutesatroomtemperatureandstoppedbyadditionof0.5ulstopsolution.The2Xassaysolutionwasaddedandthesignalwasreadafterincubationatroomtemperaturefor40minutes.

RESEARCHUSE:Forresearchuseonly,notforuseindiagnosticprocedures.

Forinformation ontheassayprinciplepleaseseethe Bridge-ItAssayTechnologyplatform pageordownloadthe protocol. Thisproductisprotectedbypatentsandpendingpatents.

SelectedCustomerPublicationsusingMediomics’Bridge-It®cAMP-PDEFluorescenceAssayKit:

1. Fermentativeproductionofalcohols. BhadraB,BhallaR,KruckebergAL,NagarajanV,PatnaikR,SuhW,inventors;ButamaxAdvancedBiofuelsLLC,assignee. US9909148B2. 2018Mar6.

2. ActivationofthePi3k/Aktpathwayandmodulationofphosphodiesteraseactivityviamembraneprogestinreceptor-alpha(mPRalpha)regulateprogestin-initiatedspermhypermotilityinAtlanticcroaker. TanW,ThomasP. BiolReprod. 2014May;90(5):105.

3. Roleofextracellularsignal-regulatedkinase5inADIpocytesignaling. ZhuH,GuarigliaS,LiW,BranchoD,WangZV,SchererPE,ChowCW. JBiolChem. 2014Feb28;289(9):6311-22.

4. TheNMRA/NMRAL1homologuePadAmodulatestheexpressionofextracellularcAMPrelaygenesduringaggregationinDictyosteliumdiscoideum. GarciandiaA,SuarezT. DevBiol. 2013Sep15;381(2):411-22.

Catalog#: PD1007-100,PD1007-384

Protocol: 

Bridge-It®PDEAssayKit,384-wellformat515.63KB

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MSDS:

Bridge-It®PDEAssayKitSDS205.92KB

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