| 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 |
1. SavilleL,SpaisC,MasonJL,AlbomMS,MurthyS,MeyerSL,AtorMA,AngelesTS,HustenJ.(2012)Time-ResolvedFluorescenceResonanceEnergyTransferasaVersatileToolintheDevelopmentofHomogeneousCellularKinaseAssays.AssayDrugDevTechnol.
2. LoMC,NgoR,DaiK,LiC,LiangL,LeeJ,EmkeyR,EksterowiczJ,VenturaM,YoungSW,XiaoSH.(2012)Developmentofatime-resolvedfluorescenceresonanceenergytransferassayforcyclin-dependentkinase4andidentificationofitsATP-noncompetitiveinhibitors.AnalBiochem,421,368.
3. PailaYD,KombrabailM,KrishnamoorthyG,ChattopadhyayA.(2011)OligomerizationoftheSEROtonin(1A)receptorinlivecells:atime-resolvedfluorescenceanisotropyapproach.JPhysChemB,115,11439.
4. MartikkalaE,Rozwandowicz-JansenA,HanninenP,Petaja-RepoU,HarmaH.(2011)Ahomogeneoussingle-labeltime-resolvedfluorescencecAMPassay.JBiomolScreen,16,356.
5. GaboritN,LarbouretC,VallagheJ,PeyrussonF,Bascoul-MolleviC,CrapezE,AzriaD,ChardesT,PoulMA,MathisG,BazinH,PelegrinA.(2011)Time-resolvedfluorescenceresonanceenergytransfer(TR-FRET)toanalyzethedisruptionofEGFR/HER2dimers:anewmethodtoevaluatetheefficiencyoftargetedtherapyusingmonoclonalantibodies.JBiolChem,286,11337.
6. LeyrisJP,RouxT,TrinquetE,VerdieP,FehrentzJA,OueslatiN,DouzonS,BourrierE,LamarqueL,GagneD,GalleyrandJC,M"KadmiC,MartinezJ,MaryS,BaneresJL,MarieJ.(2011)Homogeneoustime-resolvedfluorescence-basedassaytoscreenforligandstargetingthegrowthhormonesecretagoguereceptortype1a.AnalBiochem,408,253.
7. PosokhovYO,KyrychenkoA,LadokhinAS.(2010)Steady-stateandtime-resolvedfluorescencequenchingwithtransitionmetalionsasshort-distanceprobesforproteinconformation.AnalBiochem,407,284.
8. Alvarez-CurtoE,WardRJ,PedianiJD,MilliganG.(2010)LigandregulationofthequaternaryorganizationofcellsurfaceM3muscarinicacetylcholinereceptorsanalyzedbyfluorescenceresonanceenergytransfer(FRET)imagingandhomogeneoustime-resolvedFRET.JBiolChem,285,23318.
9. KotaS,ScampaviaL,SpicerT,BeelerAB,TakahashiV,SnyderJK,PorcoJA,HodderP,StrosbergAD.(2010)Atime-resolvedfluorescence-resonanceenergytransferassayforidentifyinginhibitorsofhepatitisCviruscoredimerization.AssayDrugDevTechnol,8,96.
10. VisserAJ,LaptenokSP,VisserNV,vanHoekA,BirchDJ,BrochonJC,BorstJW.(2010)Time-resolvedFRETfluorescencespectroscopyofvisiblefluorescentproteinpairs.EurBiophysJ,39,241.
AAT Bioquest常用产品报价单
| 产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
| AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |
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