| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 498/520 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection Histonedeacetylase(HDAC) |
| Related | MicroplateReaders BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Prepareworkingsolution:
1.1 PrepareHDAC-containingtestsamples:Dilute5–10mg/mLofHeLanuclearextractorcelllysatesat1:40inAssayBuffer(ComponentB).
Note:40µLofthedilutedsampleisenoughforonewellofa96-wellplate.Diluteextractimmediatelybeforeuse.Storethesolutiononice.
1.2 PreparedilutionsofHDACinhibitor(TrichostainA)solution:Dilute3mMTrichostatinAsolution(ComponentC)at1:100inassaybuffer(ComponentB)togeta30μMTrichostatinAsolution.Add10μLofthe30μMTrichostatinAsolutionintoeachinhibitorcontrolwell.
1.3 PrepareHDACGreen™Substrateworkingsolution:Add20µLofHDACGreen™Substrate(ComponentA)and100µLoftheSignalEnhancer(ComponentD)into5mLofAssayBuffer(ComponentB).
Note1:ThedilutedHDACGreen™Substrateworkingsolutionisnotstable,5mLofthedilutedHDACGreen™Substrateworkingsolutionisenoughfor100assays.
Note2:PreparefreshHDACGreen™Substrateworkingsolutionforeachexperiment.Keepreconstitutedworkingsolutiononiceuntiluse.
2.RunHDACAssay:
2.1 Add40μLofdilutednuclearextract,enzymesolutionorotherHDACsamplesand10μLoftestcompoundstothecorrespondingmicroplatewells(seeTable1).
Forpositivecontrol:Add40μLofdilutedHDACenzymesolutionorHeLanuclearextract(fromStep1.1)with10μLofAssayBuffer(ComponentB).
Fornegativecontrol:Add40μLofdilutedHeLanuclearextract(fromStep1.1)with10μLof30μMTrichostatinAsolution(fromStep1.2),oruseaknownsamplecontainingnoHDACactivity.
ForBlank(noEnzyme):Add50μLofAssayBuffer(ComponentB)only.
2.2 Incubatetheplateatroomtemperatureor37◦Cfor10-20minutes.
Note:ForscreeningHDACinhibitor,preincubatethecompoundswithHeLanuclearextractorpureenzymebeforeaddingHDACGreen™Substrateworkingsolution(seeStep2.3)
2.3 Add50μLofHDACGreen™Substrateworkingsolution(fromStep1.3)intoeachwell.Incubatetheplateatroomtemperatureor37oCfor30-60minutes.
2.4 MonitorfluorescenceintensityatEx/Em=490/525nm.
Table1.Layoutofnuclearextractswithtestcompoundsinasolidblack96-wellmicroplate
Samples | HeLaExtract (fromStep1.2) | AssayBuffer (ComponentB) | TrichostatinA (fromStep1.3) | TestCompounds | HDACGreen™Substrate(fromStep1.1) |
Blank(noEnzyme) | 0µL | 50µL | 0µL | 0µL | 50µL |
PositiveControl | 40µL | 10µL | 0µL | 0µL | 50µL |
NegativeControl | 40µL | 0µL | 10µL | 0µL | 50µL |
TestCompounds | 40µL | 0µL | 0µL | 10µL | 50µL |
| References&Citations |  CitationExplorer |
Marine-derivechromopeptideA,anovelclassIHDACinhibitor,suppresseshumanprostatecancercellproliferationandmigration
Authors:Jing-yaSun,Ji-dongWang,XinWang,Hong-chunLiu,Min-minZhang,Yu-ChihLiu,Chen-huaZhang,YiSu,Yan-yanShen,Yue-weiGuo
Journal:ActaPharmacologicaSinica(2017)
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Journal:Scientificreports(2016)
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