Mediomics/Bridge-It® Cyclic AMP (cAMP) Designer Assay Kit, 96-well format/122934

TheBridge-It^® CyclicAMP(cAMP)assayishighlyspecific.ATP,AMP,andcGMPhaveallbeentestedforselectivityusingthecAMPassay.NoresponsewasdetectedusingtheBridge-It^® cAMPassaywithanyofthesecompoundswithintheconcentrationrangethatwouldbeexpectedtooccurin“reallife”samples(i.e.,millimolarrangeforAMPandATP,andmicromolarrangeforcGMP).Thesensitivityandperformancecharacteristicsofthe Bridge-It^®cAMP designer andtheBridge-It^® allinone assayproductsarepresentedinthefollowingTableandFigures.

Pricing: The50-wellkitisaone-timetrialpurchase.Forbulkpricingoptions,please contactus.Foranyotherquestionsorcomments,pleasedonothesitateto contactus.Wearealwayshappytohelp!

ExampleofBridge-It®cAMPdesignerAssayforAttachedCells

TheBridge-It^® cAMP designer assayallowsforthestimulationandmeasurementofcAMPinadherentmonolayercellsattachedtothewellsofatissueculturemicroplate.Inthefollowingexample,HEK293cellsweretrypsinizedandplatedintothewellsofa96-wellpolystyrenetissueculturemicroplateatadensityof25,000cellsperwellin50µlofculturemedia.Thecellswereallowedtoattachtothebottomofthewellsovernight.Themediawasremovedthenextdayandthewellswerewashedwithabufferedsalinesolution.Thewashbufferwasthenremovedandreplacedwith50µlofKRB-IBMXbuffer.Forstimulation,1µlofforskolinatdifferentconcentrationswasaddedtoeachwellandincubatedandrotatedgentlyfor15minutesat~25ºC.ToquantifycAMPineachwell,theforskolinbuffersolutionwasremoved,100µlofthe designer AssaySolutionwasaddedtoeachwell,andthemicroplatewasincubatedfor30minutesat~25ºCwithrotation.Thewellcontentswerethentransferredintoablack96-wellmicroplateandthefluorescentsignalwasreadusingthesettingsforfluorescein(~480nmexcitation,~520nmemission).

CellPreparationandForskolinStimulation –HEK-293cellsweregrownusingstandardcellcultureconditionsto~70-80%confluency.Thecellsweretrypsinized,harvested,andwashedinserum-freeKrebsRingersBicarbonatebuffercontainingaphosphodiesteraseinhibitor(i.e.,KRB-IBMXbuffer).CyclicAMP(cAMP)levelsweremeasuredfollowingforskolinstimulationofthecellsusingtheBridge-It^® designer assay.FollowingincubationwithBridge-It^® AssaySolutionfor30minat~25°C,thefluorescentsignalwasreadat~480nmexcitation,~520nmemission.Dataanalysiswasperformedusingrelativefluorescence(RF)asasignalchangerelativetoablank(RF=(F0-F)/F0).RFanalysishasbeenshowntobehighlyreproducIBLeanddoesnotdependgreatlyontheinstrumentusedtoreadfluorescence.Examplescanbeseenbelow.

ExampleofBridge-It^® cAMP designer AssayforCellsinSUSPension

Forinformation ontheassayprinciplepleaseseethe Bridge-ItAssayTechnologyplatform pageordownloadthe protocol.

Thisproductisprotectedbypatentsandpendingpatents.

SelectedCustomerPublicationsusing Mediomics’Bridge-ItcAMP designer AssayKit:

1. TheMicrobiota-ProducedN-FormylPeptidefMLFPromotesObesity-InducedGlucoseIntolerance. WollamJ,RiopelM,XuYJ,JohnsonAMF,OfrecioJM,YingW,ElOuarratD,ChanLS,HanAW,MahmoodNA,RyanCN,LeeYS,WatrousJD,ChordiaMD,PanD,JainM,OlefskyJM. Diabetes.2019Apr22.pii:db181307.

2. StimulatoryTSH-ReceptorAntibodiesandOxidativeStressinGravesDisease.DianaT,DaiberA,OelzeM,NeumannS,OlivoPD,KanitzM,StammP,KahalyGJ. JClinEndocrinolMetab.2018Oct1;103(10):3668-3677.

3. ChronicfractalkineadmiNISTrationimprovesglucosetoleranceandpancreaticendocrinefunction.RiopelM,SeoJB,BandyopadhyayGK,LiP,WollamJ,ChungH,JungSR,MurphyA,WilsonM,deJongR,PatelS,BalakrishnaD,BilakovicsJ,FanjulA,PlonowskiA,KohDS,LarsonCJ,OlefskyJM,LeeYS. JClinInvest.2018Apr2;128(4):1458-1470.

4. ToxicityofCry1AtoxinsfromBacillusthuringiensistoCF1cellsdoesnotinvolveactivationofadenylatecyclase/PKAsignalingpathway.PortugalL,Muñóz-GarayC,MartínezdeCastroDL,SoberónM,BravoA. InsectBiochemMolBiol.2017Jan;80:21-31.

5. Pannexin3isrequiredfornormalprogressionofskeletaldevelopmentinvertebrates.OhSK,ShinJO,BaekJI,LeeJ,BaeJW,AnkamerddyH,KimMJ,HuhTL,RyooZY,KimUK,BokJ,LeeKY. FASEBJ.2015Nov;29(11):4473-84.

Thisproductisprotectedby patentsandpendingpatents.

Catalog#: 122934(50measurements), 122935(96measurements)

Protocol: 

Bridge-It®cAMPDesignerAssays,96-wellformat792.76KB

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SDS: 

Bridge-It®cAMPdesignerassaykitSDS210.98KB

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