| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 780/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | R/L |
| Category | ProteinBiochemistry Generalproteins |
| Related | BiochemicalAssays |
Uponreceipt,storeMaleimideBlue™(ComponentA)at-20°C(preferat-80°C),keptfromlightandmoisture.Whenstoredproperly,thekitcomponentsshouldbestableforsixmonths.
Note1:DonotfreezeSpinColumn(ComponentC).
Note2:Warmallthecomponentsbeforeruntherequiredassays.50to100µgmaleimide-linkedantibodyorproteinsampleisneededfordeterminingtheamountofmaleimide.
1.Preparesamplesolution:
1.1Use50to100µgmaleimidesample(proteinorotherpolymers).
1.2Adjustthevolumeto100µLwithAssayBuffer(ComponentB).
Note:Themaleimide-linkedantibodyorproteinsampleshouldbeinpH=6.0bufferandwithoutfreemaleimide.
2.RunMaleimideAssay:
2.1 Addthemaleimidesample(fromStep1.2)toonevialofMaleimideBlue™(ComponentA).
2.2 Mixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
2.3 Keepthereactionmixtureatroomtemperatureandrotateorshakefor30-60minutes.
3.PrepareSpinColumnforSamplePurification:
3.1 InverttheSpinColumn(ComponentC)severaltimestoresUSPendthesettledgelandremoveanybubbles.
3.2 SnapoffthetipandplacethecolumnintheWashingTube(2mL,ComponentD).Removethecaptoallowtheexcesspackingbuffertodrainbygravitytothetopofthegelbed.Ifcolumndoesnotbegintoflow,pushcapbackintocolumnandremoveitagaintostarttheflow.Discardthedrainedbuffer,andthenplacethecolumnbackintotheWashingTube.However,centrifugeimmediatelyifthecolumnisplacedintoa12x75mmtesttube(notprovided).
3.3 Centrifugefor1mininaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
3.4 Apply1mLAssayBuffer(ComponentB)tothecolumn,letthebufferdrainoutbygravity,orcentrifugethecolumnfor1mintoremovethebuffer.Discardthebufferfromthecollectiontube.Repeatthisprocessfor3-4times.
3.5 Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethereactionbuffer.Discardthebuffer.
Note1:SpinColumn(ComponentC)canfitinto2mLmicrocentrifugetubesor12x75mmtesttubesforsamplecollectionduringcentrifugation.Usethe2mLmicrotubesprovidedwiththecolumnsfortheinitialcolumnequilibrationstep.
Note2.Swingingbucketcentrifugescapableofgeneratingaminimumforceof1,000xgaresuitableforBio-Spincolumnuse.Thegravitationalforcecreatedataparticularrevolutionspeedisafunctionoftheradiusofthemicrocentrifugerotor.Consulttheswingingbucketcentrifugeinstructionmanualfortheinformationaboutconversionfromrevolutionsperminute(RPM)tocentrifugalorg-force.Alternatively,usetheequation(1)tocalculatethespeedinRPMrequiredtoreachthegravitationalforceof1,000xg.
RCF(g)=(1.12x10-5)x(RPM)2xr (1)
RCF=therelativecentrifugalforce,
RPM=thespeedoftherotor
r=theradiusincentimetersmeasuredfromthecenteroftherotortothemiddleoftheBio-Spincolumn
4.PurifyMalemideReactionProduct:
4.1 Placethecolumn(fromStep3.5)inacleanCollectingTube(1.5mL,ComponentE).Carefullyloadthesample(100μL)directlytothecenterofthecolumn.
4.2 Afterloadingthesample,add10μLAssayBuffer(ComponentB)tothetopandcentrifugethecolumnfor5minat1,000xg,andcollectthesolutionintothecollectingtube.
5.RunAbsorptionSpectrawith0.2mLor0.5mLQuartzCuvette
5.1 Dilutethemaleimidereactionproduct(fromStep4.2)by5-10foldswithAssayBuffer(ComponentB)dependingonthecuvettesizeusedandtheabsorbancereading.
Note:Thedilutionfactordoesn’taffectthefinalmaleimidequantitationresult.
5.2 Measuretheabsorptionspectrumfrom900nmto250nmrange,oronlyreadtheabsorbancenumberat280nmand782nm.
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