| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 534/619 |
| MW | 1565.59 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | EnzymeDetection PeptidasesandProteases |
| Related | CellApoptosis ApoptosisandCytotoxicity BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1. GeneralSolutionCaspaseAssaysUsingAMC,AFC,pNA,R110andProRedSubstrates
1.1. Preparea10mMstocksolutioninDMSO.
1.2. Preparea2Xcaspase substrate(50µM)assaysolutionasthefollowing:
50µLsubstratestocksolution
100 µLDTT(1M)
400 µLEDTA(100mM)
10mLTrisBuffer(20mM),pH=7.4
1.3. Mixequalvolumeofthecaspasestandardsorsampleswith2Xcaspasesubstrateassaysolution(fromStep1.2),andincubatethesolutionsatroomtemperatureforatleast1hour.
1.4. Monitorthefluorescenceusingafluorescencemicroplatereader,orabsorbanceusinganabsorbancemicroplatereader.
2. CellCaspaseAssaysUsingCell-PermeableFMKCaspaseProbes
2.1. Preparea2-5mMstocksolutioninDMSO.
2.2. Treatcellsasdesired.
2.3. Preparea2Xpermeablecaspasesubstrate(20µM)assaysolutionbydilutingtheDMSOstocksolution(fromStep2.1)inHankswith20mMHepesbuffer(HHBS).
2.4. Mixequalvolumeofthetreatedcellswith2Xcaspasesubstrateassaysolution(fromStep2.3),andincubatethecellsina37°C,5%CO2incubatorforatleast1hour.
2.5. WashthecellswithHHBSforatleastonce.
2.6. Monitorthefluorescenceintensitybyaflowcytometer,afluorescencemicroscopeorafluorescencemicroplatereader.
3. CellCaspaseAssaysUsingCell-PermeableFMKCaspaseProbes(For#13470-13476only)
3.1. Preparea250Xstocksolutionbyadding50µLDMSOintothevial.
3.2. Treatcellsasdesired.
3.3. Add250XDMSOstocksolution(fromStep3.1)intothecellsolutionata1:250ratio(suchas2uLto500uLcells),andincubatethecellsina37°C,5%CO2incubatorfor1hour.
3.4. WashthecellswithHHBSforatleastonce.
3.5. Monitorthefluorescenceintensitybyflowcytometer,fluorescencemicroscopyorfluorescentmicroplatereader.
| References&Citations |  CitationExplorer |
Degenerinchannelactivationcausescaspase-mediatedproteindegradationandmitochondrialdysfunctioninadultC.elegansmuscle
Authors:ChristopherJGaffney,FreyaShephard,JeffChu,DavidLBaillie,AnnRose,DumitruConstantin-Teodosiu,PaulLGreenhaff,NathanielJSzewczyk
Journal:JournalofCachexia,SarcopeniaandMuscle(2015)
