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AAT Bioquest/Amplite™ Fluorimetric Renin Assay Kit *Red Fluorescence*/13530/100 tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 545/576 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection PeptidasesandProteases |
| Related | BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1. PrepareRenincontainingBIOLOGicalsamplesasdesired.
2.PrepareReninAssayMixturebydilutereconstituted100XReninRed™Substratestocksolution(ComponentA)with
AssayBuffer(ComponentC)at1:100asshowninTable1.
Table1:ReninAssayMixtureforone96-wellplate(100assays)
Components | Volume |
ReninRed™Substrate(ComponentA)* | 50µL |
AssayBuffer(ComponentC) | 5mL |
Totalvolume | 5.05mL |
*Note:TheReninRed™Substrateshouldbeusedpromptly.Anyremainingsolutionshouldbealiquotedandfrozenat-20°C.
3.PrepareseriallydilutedReninstandards(0to1µg/mL):
3.1.Add12.5μLof40µg/mLReninStandard(ComponentB)into487.5μLofAssayBuffer(ComponentB)toget1µg
/mLReninstandardsolution.
3.2.Take150μLof1µg/mLReninstandardsolution(fromStep4.1)toperform1:3serialdilutionstoget300,100,30,
10,3,1and0ng/mLseriallydilutedReninstandards.
3.1.AddReninstandardsand/orRenin-containingtestsamplesintoablackwall/solidbottom96-wellmicroplateas
describedinTables2and3.
Table2.LayoutofReninstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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Ren1 | Ren1 | …. | …. | …. | …. |
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Ren2 | Ren2 |
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Ren3 | Ren3 |
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Ren4 | Ren4 |
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Ren5 | Ren5 |
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Ren6 | Ren6 |
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Ren7 | Ren7 |
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Note:Ren=ReninStandards,BL=Blankcontrol,TS=testsamples.
Table3.Reagentcompositionforeachwell
ReninStandard | BlankControl | TestSample |
SerialDilutions*(50μL) | AssayBuffer(ComponentB):50μL | 50μL |
Note1:AddtheseriallydilutedReninstandardsfrom1ng/mLto1000ng/mLintoeachwellfromRen1toRen7induplicate.
For384-wellplates,use25μL/well.
Note2:TheReninstandardsareforpositivecontrolonly,andshouldnotbereliedonasaquantitationstandardforenzymeactivity.
4.Runtheenzymereaction:
4.1.Pre-incubatetheplateatadesiredtemperaturefortheenzymereaction(e.g.25°Cor37°C)for10-15minifyouare
screeningRenininhibitors.
4.2.Add50μL(96-well)or25μL(384-well)ofReninRed™substratesolution(fromStep3)tothesampleandcontrol
wellsoftheassayplate.
4.3. Incubatethereactionat37°Cincubatorfor30to60minutes.
4.4. MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=540/590nm(cutoff=570mn).
Forkineticreading:Immediatelystartmeasuringfluorescenceintensityandcontinuouslyrecorddataevery5minutesfor30to60minutes.
Forend-pointreading:Incubatethereactionat37°Cfor60minutesorlonger,keptfromlightifpossible.Andthenmeasurethefluorescenceintensity.
Note:Theselectivityofthereninsubstrateusedinthekithasnotbeenthoroughlytested.Itmayalsoresponsetootherproteasessincepeptide-basedproteasesubstratesgenerallyhavelowselectivity. Onemightusearenin-specificinhibitorforitsspecifictest,suchasin thepresenceofarenin-specificinhibitor,hydrolysisofthesubstrateisonlyduetothenon-specificproteaseactivity.ThedifferencebetweenthetotalactivityandtheactivityinthepresenceofreninspecificInhibitor,givesthereninactivityinthesample.
| References&Citations |  PrinterFriendlyVersion |
1. DeMelloWC,GerenaY.(2009)Prolongedexposureofcardiaccellstoreninplusangiotensinogenreducesintracellularrenininthefailingheart.OntheroleofangiotensinII-AT1complexinternalization.RegulPept,155,139.
2. FriisUG,MadsenK,SvenningsenP,HansenPB,GulaveerasingamA,JorgensenF,AalkjaerC,SkottO,JensenBL.(2009)Hypotonicity-inducedReninexocytosisfromjuxtaglomerularcellsrequiresaquaporin-1andcyclooxygenase-2.JAmSocNephrol,20,2154.
3. SchmiedtCW,HurleyKA,TongX,RakhmanovaVA,PoCL,HurleyDJ.(2009)Measurementofplasmareninconcentrationincatsbyuseofafluorescenceresonanceenergytransferpeptidesubstrateofrenin.AmJVetRes,70,1315.
4. VargasSL,TomaI,KangJJ,MeerEJ,Peti-PeterdiJ.(2009)ActivationofthesuccinatereceptorGPR91inmaculadensacellscausesreninrelease.JAmSocNephrol,20,1002.
5. WankaH,KesslerN,EllmerJ,EndlichN,PetersBS,ClausmeyerS,PetersJ.(2009)CytosolicreninistargetedtomitochondriaandinducesapoptosisinH9c2ratcardiomyoblasts.JCellMolMed,13,2926.
6. DuD,KatoT,NABIAH,SuzukiF,ParkEY.(2008)Expressionoffunctionalhuman(pro)reninreceptorinsilkworm(Bombyxmori)larvaeusingBmMNPVbacmid.BiotechnolApplBiochem,49,195.
7. FeldtS,BatenburgWW,MazakI,MaschkeU,WellnerM,KvakanH,DechendR,FiebelerA,BurckleC,ContrepasA,JanDanserAH,BaderM,NguyenG,LuftFC,MullerDN.(2008)Proreninandrenin-inducedextracellularsignal-regulatedkinase1/2activationinmonocytesisnotblockedbyaliskirenorthehandle-regionpeptide.Hypertension,51,682.
8. GlennST,JonesCA,PanL,GrossKW.(2008)Invivoanalysisofkeyelementswithinthereninregulatoryregion.PhysiolGenomics,35,243.
9. HannerF,vonMaltzahnJ,MaxeinerS,TomaI,SiposA,KrugerO,WilleckeK,Peti-PeterdiJ.(2008)Connexin45isexpressedinthejuxtaglomerularapparatusandisinvolvedintheregulationofreninsecretionandbloodpressure.AmJPhysiolRegulIntegrCompPhysiol,295,R371.
10. ImanishiT,TsujiokaH,IkejimaH,KuroiA,TakaradaS,KitabataH,TanimotoT,MuragakiY,MochizukiS,GotoM,YoshidaK,AkasakaT.(2008)Renininhibitoraliskirenimprovesimpairednitricoxidebioavailabilityandprotectsagainstatheroscleroticchanges.Hypertension,52,563.
