Overview | PrinterFriendlyVersion |
Ex/Em(nm) | 492/516 |
MW | N/A |
CAS# | N/A |
Solvent | DMSO |
Storage | F/D/L |
Category | SuperiorLabelingDyes iFluor™DyesandKits |
Related | Generalproteins LabelingviaAminoGroups BiochemicalAssays |
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.
References&Citations | CitationExplorer |
Roleofreactiveoxygenspecies-mediatedMAPKandNF-$kappa$Bactivationinpolygonatumcyrtonemalectin-inducedapoptosisandautophagyinhumanlungadenocarcinomaA549cells
Authors:TaoLiu,LeiWu,DiWang,HaiyangWang,JinwuChen,ChunlanYang,JinkuBao,ChuanfangWu
Journal:JournalofBiochemistry(2016):mvw040
Determinationofsodiumbenzoateinfoodproductsbyfluorescencepolarizationimmunoassay
Authors:LinlinRen,MengMeng,PengWang,ZhihuanXu,SergeiAEremin,JunhongZhao,YongmeiYin,RimoXi
Journal:Talanta(2014):136--143
ComplexescontainingcationicandanionicpH-sensitiveliposomes:comparativestudyoffactorsinfluencingplasmidDNAgenedeliverytotumors
Authors:YanChen,JiSun,YingLu,ChunTao,JingbinHuang,HeZhang,YuanYu,HaoZou,JingGao,YanqiangZhong
Journal:Internationaljournalofnanomedicine(2013):1573
SwimmingbehaviorofthemonotrichousbacteriumPseudomonasfluorescensSBW25
Authors:LiyanPing,JanBirkenbeil,ShamciMonajembashi
Journal:FEMSmicroBIOLOGyecology(2013):36--44