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AAT Bioquest/Amplite™ Colorimetric Maleimide Quantitation Kit/5525/100 tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 324/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | ProteinBiochemistry Generalproteins |
| Related | BiochemicalAssays |
Note:Thawallthekitcomponentsatroomtemperaturebeforestartingtheexperiment.
1.Prepare500XMEAstocksolution:Add0.2mLdistilledwaterintoMEA(ComponentA)
Note:10µLofthe500XMEAstocksolutionisenoughfor50reactions(0.5mL/reaction).Theunused500XMEAstocksolutionshouldbedividedintosingleusealiquots,storedat-20oCandkeptfromlight.
2.PrepareMEAworkingsolution:Add10µLofthe500XMEAstocksolution(fromStep1)into5mLdistilledwater.
Note:TheMEAworkingsolutionisnotstable.Preparefreshbeforeuse(lessthan2hoursatroomtemperature).
3.Prepare50X4,4’-DTDPstocksolution:Add1mLDMSO(ComponentD)intothevialof4,4’-DTDP(ComponentB)tohave50X4,4’-DTDPstocksolution.
Note:100µLofthe50X4,4’-DTDPstocksolutionisenoughfor10reactions(0.5mL/reaction).Theunused50X4,4’-DTDPstocksolutionshouldbedividedintosingleusealiquots,storedat-20oCandkeptfromlight.
4.RunMaleimideassay:
4.1 Setup3TotalSHtubes:Add0.4mLofAssayBuffer(ComponentC)and0.1mLofMEAworkingsolution(fromStep2)intoeachtubeandincubateatroomtemperaturefor20minutes.
4.2 Setup3testtubesforeachsample:Add0.05mgoftestsampleandsufficientAssayBuffer(ComponentC)tomakethetotalvolumeof0.4mL/tube.Add0.1mLofMEAworkingsolution(fromStep2)intoeachtubeandincubateatroomtemperaturefor20minutes.
4.3 MeasuretheabsorbanceoftheAssayBuffer(ComponentC)astheblankcontrolat324nm.
4.4 ProceedtoTotalSHdeterminationwhiletubesarestillincubating(fromStep4.1).Add10μLof50X4,4’-DTDPstocksolution(fromStep3)intoeachTotalSHtubeandincubateatroomtemperaturefor2min.
Note:Donotadd50X4,4’-DTDPstocksolutiontothesamplecontainingtubesyet.
4.5 Measuretheabsorbanceofthe3TotalSHtubesat324nmwithoutwashingthecuvette.RecordthereADIngsandaveragethemtohave"ODTSH".
4.6 Cleanthecuvetteandreadtheabsorbanceofthefirstsampletube(fromStep4.2)at324nm(OD0)beforeaddany50X4,4’-DTDPstocksolution.
4.7 Add10μLof50X4,4’-DTDPstocksolution(fromStep3)intothesamplecuvette(fromStep4.6)andmixwell.Incubatethesampleatroomtemperaturefor2minutesandreadtheabsorbanceat324nm(OD).
4.8 Cleanthecuvette,andrepeatSteps4.6and4.7fortheremainingtubes.Recordallreadings.
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