| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 346/617 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | SuperiorLabelingDyes trFluor™DyesandKits |
| Related | BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.Prepareproteinsolution(SolutionA):
Forlabeling50µgprotein(assumingthetargetproteinconcentrationis1mg/mL),mix1.5µL(3%ofthetotalreactionvolume)ofReactionBuffer(ComponentB)with50µLofthetargetproteinsolution.
Note1.Ifyouhaveadifferenceproteinconcentration,adjusttheproteinvolumeaccordinglytomake~50µgproteinavailableforyourlabelingreaction.
Note2:Forlabeling100µgprotein(assumingthetargetproteinconcentrationis1mg/mL),mix3µL(3%ofthetotalreactionvolume)ofReactionBuffer(ComponentB)with100µLofthetargetproteinsolution.
Note3:Theproteinshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4;Iftheproteinisdissolvedinglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,oruseAmiconUltra-0.5,Ultracel-10Membrane,10 kDa(cat#UFC501008fromMillipore)toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforproteinprecipitation.
Note4:Impureantibodiesorantibodiesstabilizedwithbovineserumalbumin(BSA)orgelatinwillnotbelabeledwell.
Note5:Theconjugationefficiencyissignificantlyreducediftheproteinconcentrationislessthan1mg/mL.Foroptimallabelingefficiencythefinalproteinconcentrationrangeof1-2mg/mLisrecommended.
2.Runconjugationreaction:
2.1 Addtheproteinsolution(SolutionA)toONEvialoflabelingdye(ComponentA),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
Note:Usebothvials(ComponentA)oflabelingdyetolabel100µgproteinbydividingthe100µgproteininto2x50µgproteinandreactingeach50µgproteinwithonevialoflabelingdye.Combinetwovialsforthenextstep.
2.2 Keeptheconjugationreactionmixtureatroomtemperaturefor30-60minutes.
Note:Theconjugationreactionmixturecanberotatedorshakenforlongertimeifdesired.
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 Apply1-2mL1XPBS(pH7.2-7.4)tothecolumn.AftereachapplicationofPBS,letthebufferdrainoutbygravity,orcentrifugethecolumnfor1mintoremovethebuffer.Discardthebufferfromthecollectiontube.Repeatthisprocessfor3-4times.
3.5 Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
4.Purifytheconjugation:
4.1 Placethecolumn(fromStep3.5)inacleanCollectingTube(1.5mL,ComponentE).Carefullyloadthesample(50–100μL)directlytothecenterofthecolumn.
4.2 Afterloadingthesample,add1XPBS(pH7.2-7.4)tomakethetotalvolumeof110μL.Centrifugethecolumnfor5minat1,000xg,andcollectthesolutionthatcontainsthedesireddye-labeledprotein.
| References&Citations |  PrinterFriendlyVersion |
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