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AAT Bioquest/Amplite™ Universal Fluorimetric MMP Activity Assay Kit *Red Fluorescence*/13511/100 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 545/576 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | EnzymeDetection PeptidasesandProteases |
| Related | MicroplateReaders BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareMMPscontainingBIOLOGicalsamplesasdesired.
2.Activatepro-MMPs:
2.1 Make2mMAPMAworkingsolution(2X):Dilute1MAPMA(ComponentB)withAssayBuffer(ComponentC)at1:500to2mMAPMAworkingsolution(2X).
Note:APMAbelongstoorganicmercury.Handlewithcare!Disposeitaccordingtolocalregulations.
2.2 IncubateMMPswithAPMA:IncubatetheMMPcontaining-samplesorpurifiedMMPswithequalvolumeof2mMAPMAworkingsolution(2X,fromStep2.1).RefertoAppendixIforincubationtime.ActivateMMPimmediatelybeforetheexperiment.
Note1:Keepenzyme-containingsamplesonice.Avoidvigorouslyvortexingtheenzyme.Prolongedstorageoftheactivatedenzymewilldeactivatetheenzyme.
Note2:Forenzymeactivation,itispreferablyactivatedathigherproteinconcentration.Afteractivation,youmayfurtherdilutetheenzyme.
3.Prepareworkingsolutions:
3.1 MakeMMPRed™substrateworkingsolution:DiluteMMPRed™Substratesolution(ComponentA)withAssayBuffer(ComponentC)at1:100asshowninTable1.
Table1.MMPRed™substrateworkingsolutionforone96-wellplate(100assays)
Components | Volume |
MMPRed™Substrate(ComponentA) | 50µL |
AssayBuffer(ComponentC) | 5mL |
Totalvolume | 5mL |
3.2 MakeMMPdilution:DiluteMMPstoanappropriateconcentrationwithAssayBuffer(ComponentC)ifpurifiedMMPisused.
Note:Pro-MMPneedstobeactivatedbeforeuse(seeStep2.2).Avoidvigorouslyvortexingtheenzyme.
3.3 Makeinhibitorsandcompoundsdilution:MakedilutionsofknownMMPsinhibitorsandtestcompoundsasdesiredifyouarescreeningMMPsinhibitors.
4.Setuptheenzymaticreactionina96-wellmicroplateaccordingtoTable2andTable3.
Table2.Layoutoftheappropriatecontrols(asdesired)andtestsamplesina96-wellmicroplate
SC | SC |
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IC | IC |
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VC | VC |
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TC | TC |
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TS | TS |
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…. | …. |
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…. | …. |
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Note:SC=SubstrateControl,IC=InhibitorControl,VC=VehicleControl,TC=TestCompoundControl,
TS=TestSamples.
Table3.Reagentcompositionforeachwell
SubstrateControl | InhibitorControl | VehicleControl | TestCompoundControl* | TestSample |
Assaybuffer
| MMPdilutionandknownMMPsinhibitor | MMPdilutionandvehicleusedtodelivertestcompound | Assaybufferandtestcompound | MMPdilutionwithtestcompound |
Totalvolume:50μL | 50μL | 50μL | 50μL | 50μL |
Note1:*Somestronglyfluorescenttestcompoundsmayresultinfalse-positiveresults.
Note2:Makethetotalvolumeofallthecontrolsto50μLfora96-wellplateor20μLfora384-wellplatebyusingAssayBuffer(ComponentC).
5.Runtheenzymereaction:
5.1 Pre-incubatetheplateatadesiredtemperaturefortheenzymereaction(e.g.25°Cor37°C)for10-15minifyouarescreeningMMPsinhibitors.
5.2 Add50µL(96-well)or20µL(384-well)ofMMPRed™substrateworkingsolution(fromStep3.1)tothesampleandcontrolwellsoftheassayplate.Mixthereagentswell.
5.3 MonitorthefluorescenceintensitywithafluorescenceplatereaderatEx/Em=540/590nm.
ForkineticreADIng:Immediatelystartmeasuringfluorescenceintensityandcontinuouslyrecorddataevery5minutesfor30to60minutes.
Forend-pointreading:Incubatethereactionatroomtemperaturefor30to60minutes,keptfromlightifpossible.Mixthereagentswell,andthenmeasurethefluorescenceintensity.
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