| Ex/Em(nm) | 498/520 |
| MW | 1155.30 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | EnzymeDetection
PeptidasesandProteases
|
| Related | BiochemicalAssays
|
Thenon-fluorescentR110substratesgeneratethebrightgreenfluorescentrhodamine110productthathasEx/Em=494/521nm,andcanbeeasilydetectedwithaFITCfilterset.Ingeneral,R110substratesaremuchmoresensitivethantheAMC-,AFC-or4-nitroaniline-basedsubstrates.ThisR110substrateisusedformonitoringtheproteaseactivitiesoftheproteasome.Themostcommonformoftheproteasomeisknownasthe26Sproteasomethatcontainsone20Scoreparticlestructureandtwo19Sregulatorycaps.All20Sparticlesconsistoffourstackedheptamericringstructuresthatarethemselvescomposedoftwodifferenttypesofsubunits;alphasubunitsarestructuralinnature,whereasbetasubunitsarepredominantlycatalytic.Theoutertworingsinthestackconsistofsevenalphasubunitseach,whichserveasdockingdomainsfortheregulatoryparticlesandthealphasubunitsN-terminiformagatethatblocksunregulatedaccessofsubstratestotheinteriorcavity.Theinnertworingseachconsistofsevenbetasubunitsandcontaintheproteaseactivesitesthatperformtheproteolysisreactions.AATBioquestoffersagroupofR110substratesformonitoringtheproteaseactivitiesoftheproteasomeatdifferentsubsites,i.e.,(i)sub-sites:beta1c,Z-LLE-R110;beta2c,Ac-KQL-R110;beta5c,Ac-WLA-R110;beta1i,Ac-PAL-R110;beta2i,Ac-KQL-R110;beta5c,Ac-WLA-R110andSuc-LLVY-R110;andbeta5i,Ac-ANW-R110.TheproteaseactivityismeasuredbymonitoringtheR110liberationovertimeusingexcitationandemissionwavelengthsof490nmand520nmrespectively.Movemouseovergridtodisplaywavelength&intensityvalues.
Wavelength(nm)
1. Makea10mMstocksolutionbyadding85µLofDMSOintothevialof1mg(AC-DEVD)2-R110.
2. Preparea20-40uMof 2Xproteasomeassaysolutionin25mMHEPESbuffer pH7.5,containing 5mMEDTA,2mMDTT,and 0.2%TritonX-100.
3. Mixequalvolumeoftheproteasomestandardsorsampleswith2Xproteasomeassaysolution,andincubateatroomtemperatureforatleast1hour.
4. MonitorthefluorescenceincreaseatEx/Em=490/525nm.
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