| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 490/515 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | ProteinBiochemistry Generalproteins |
| Related | BiochemicalAssays |
1.Prepare500XMaleimideGreen™stocksolution:
Add20µLofDMSO(ComponentE)intotheMaleimideGreenvial(ComponentA)tomake500Xstocksolution.
Note:10µLofthestocksolutionisenoughforone96-wellplate.TheunusedMaleimideGreen™stocksolutionshouldbedividedintosingleusealiquots,storedat-20oCandkeptfromlight.
2.Prepare20Xmaleimidereactionmixture:
Add10µLof500XMaleimideGreen™stocksolution(fromStep1)into250µLReactionBuffer(fromComponentB),andmixthemwell.Incubatethe20Xmaleimidereactionmixtureatroomtemperaturefor30min,protectedfromlight.
Note1:Itisveryimportanttoincubatethe20Xmaleimidereactionmixtureatroomtemperatureforatleast30mintomaximizethesignaltobackgroundratio.
Note2:YoushouldseetheyellowcolorafteraddingtheMaleimideGreen™stocksolutionintoreactionbuffer.
3.Preparemaleimideassaymixture:
Addthewholecontentsof20Xmaleimidereactionmixture(260µLfromStep2)into5mLofassaybuffer(ComponentC),andmixwell.
Note:Thismaleimideassaymixtureisnotstable.Usewithin1hour.
4.PrepareserialdilutionsofN-ethylaleimidestandard(0to10μM):
4.1 Add10μLof10mM(10nmol/µL)N-ethylmaleimidestandardstocksolution(ComponentD)to990µLofassaybuffer(ComponentC)togenerate100µM(100pmol/µL)N-ethylmaleimidestandardsolution.
Note:Theunused10mMN-ethylmaleimidestandardsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
4.2 Take200μLof100μMN-ethylmaleimidestandardsolution(fromStep4.1)toperform1:3serialdilutionstoget30,10,3,1,0.3,0.1and0μMserialdilutionsofN-ethylmaleimidestandard.
4.3 AddN-ethylmaleimidestandardsandmaleimide-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2
Table1.LayoutofN-ethylmaleimidestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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MS1 | MS1 | …. | …. | …. | …. |
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MS2 | MS2 |
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MS3 | MS3 |
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MS4 | MS4 |
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MS5 | MS5 |
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MS6 | MS6 |
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MS7 | MS7 |
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Note:MS=N-ethylmaleimideStandards,BL=BlankControl,TS=TestSamples.
Table2.Reagentcompositionforeachwell
N-ethylmaleimideStandard | BlankControl | TestSample |
SerialDilutions*(50μL) | AssayBuffer:50μL | 50μL |
*Note:AddtheserialdilutionsofN-ethylmaleimidestandardfrom0.1μMto100μMintowellsfromMS1toMS7induplicate.
5.Runmaleimideassay:
5.1 Add50μLofmaleimideassaymixture(fromStep3)toeachwelloftheN-ethylmaleimidestandard,blankcontrol,andtestsamples(seeStep4.3)tohavethetotalmaleimideassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofmaleimidereactionmixtureintoeachwell.
5.2 Incubatethereactionmixturefor5to30minutesatroomtemperature,protectedfromlight.
Note:Forbestresults,thefluorescenceintensityshouldbereadwithin30minutesduetothefactthatthefluorescencebackgroundincreaseswithtime.
5.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=490/520nm.
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