| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 649/665 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | SuperiorLabelingDyes iFluor™DyesandKits |
| Related | Generalproteins LabelingviaAminoGroups |
| Spectrum | AdvancedSpectrumViewer |
- Prepareproteinsolution(SolutionA):
Forlabeling50ugprotein(assumingthetargetproteinconcentrationis1mg/mL),mix5μL(10%ofthetotalreactionvolume)ofReactionBuffer(ComponentB)with50uLofthetargetproteinsolution.
Note1:Ifyouhaveadifferenceproteinconcentration,adjusttheproteinvolumeaccordinglytomake~50µgproteinavailableforyourlabelingreaction.
Note2:Forlabeling100ugprotein(assumingthetargetproteinconcentrationis1mg/mL),mix10uL(10%ofthetotalreactionvolume)ofReactionBuffer(ComponentB)with100uLofthetargetproteinsolution.
Note3:Theproteinshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4;Iftheproteinisdissolvedinglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,oruseAmiconUltra-0.5,Ultracel-10Membrane,10kDa(cat#UFC501008fromMillipore)toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforproteinprecipitation.
Note4:ImpureantibodiesorantibodiesstABIlizedwithbovineserumalbumin(BSA)orgelatinwillnotbelabeledwell.
Note5:Theconjugationefficiencyissignificantlyreducediftheproteinconcentrationislessthan1mg/mL.Foroptimallabelingefficiencythefinalproteinconcentrationrangeof1-2mg/mLisrecommended. - Runconjugationreaction::
- Addtheproteinsolution(SolutionA)toONEvialoflabelingdye(ComponentA),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
Note:Usebothvials(ComponentA)oflabelingdyetolabel100ugproteinbydividingthe100ugproteininto2x50ugproteinandreactingeach50ugproteinwithonevialoflabelingdye.Combinetwovialsforthenextstep. - Keeptheconjugationreactionmixtureatroomtemperaturefor30-60minutes.
Note:Theconjugationreactionmixturecanberotatedorshakenforlongertimeifdesired.
- Addtheproteinsolution(SolutionA)toONEvialoflabelingdye(ComponentA),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
- StopConjugationreaction:
- Add5uL(for50ugprotein)or10uL(for100ugprotein)whichis10%ofthetotalreactionvolumeofTQ-DyedQuenchBuffer(ComponentC)intotheconjugationreactionmixture(fromstep2.2),mixthemwell.
- Incubateatroomtemperaturefor10minutes.
- Thelabeledprotein(antibody)isnowreadytouse.
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