Overview | PrinterFriendlyVersion |
Ex/Em(nm) | 531/556 |
MW | 892.10 |
CAS# | N/A |
Solvent | DMSO |
Storage | F/D/L |
Category | SuperiorLabelingDyes iFluor™DyesandKits |
Related | Amine-ReactiveProbes LabelingviaThiolGroups |
Spectrum | AdvancedSpectrumViewer |
- Prepareproteinstocksolution(SolutionA):
- (Optional)ifyourproteindoesnotcontainafreecysteine,youmusttreatyourproteinwithDTTorTCEPtogenerateathiolgroup.10molarequivalentsofDTTorTCEParesufficientforconvertingadisulfidebondtotwofreethiolgroups.IfDTTisusedyoumustremovefreeDTTbydialysisorgelfiltrationbeforeconjugatingadyemaleimidetoyourprotein.Followingisasampleprotocolforgeneratingafreethiolgroup:
a.Prepareafreshsolutionof1MDTT(15.4mg/100µl)indistilledwater.
b.MakeIgGsolutionin20mMDTT:add20µlofDTTstockpermlofIgGsolutionwhilemixing.Letstandatroomtempfor30minuteswithoutadditionalmixing(tominimizereoxidationofcysteinestocystines).
c.PassthereducedIgGoverafiltrationcolumnpre-equilibratedwith"ExchangeBuffer".Collect0.25mlfractionsoffthecolumn.
d.DeterminetheproteinconcentrationsandpoolthefractionswiththemajorityoftheIgG.Thiscanbedoneeitherspectrophotometricallyorcolorimetrically.
e.Carryouttheconjugationassoonaspossibleafterthisstep(seebelow).
Note1:IgGsolutionsshouldbe>4mg/mlforthebestresults.Theantibodyshouldbeconcentratediflessthan2mg/ml.Includeanextra10%forlossesonthebufferexchangecolumn.
Note2:ThereductioncanbecarriedoutinalmostanybuffersfrompH6to7,e.g.,MES,phosphateorTRISbuffers.
Note3:Stepscanddcanbereplacedbydialysis. - Mix100µLofareactionbuffer(e.g.,100mMMESbufferwithpH~6.0)with900µLofthetargetproteinsolution(e.g.antibody,proteinconcentration>2mg/mlifpossible)togive1mLproteinlabelingstocksolution.
Note1:ThepHoftheproteinsolution(SolutionA)shouldbe6.5±0.5.
Note2:ImpureantibodiesorantibodiesstABIlizedwithbovineserumalbumin(BSA)orotherproteinswillnotbelabeledwell.
Note3:Theconjugationefficiencyissignificantlyreducediftheproteinconcentrationislessthan2mg/mL.Foroptimallabelingefficiencythefinalproteinconcentrationrangeof2-10mg/mLisrecommended.
- (Optional)ifyourproteindoesnotcontainafreecysteine,youmusttreatyourproteinwithDTTorTCEPtogenerateathiolgroup.10molarequivalentsofDTTorTCEParesufficientforconvertingadisulfidebondtotwofreethiolgroups.IfDTTisusedyoumustremovefreeDTTbydialysisorgelfiltrationbeforeconjugatingadyemaleimidetoyourprotein.Followingisasampleprotocolforgeneratingafreethiolgroup:
- Preparedyestocksolution(SolutionB):
AddanhydrousDMSOintothevialofiFluor™dyemaleimidetomakea10-20mMstocksolution.MixwellbypipettingorvortexundersuBDuedlight(ifpossible).
Note:Preparethedyestocksolution(SolutionB)beforestartingtheconjugation.Usepromptly.Extendedstorageofthedyestocksolutionmayreducethedyeactivity.SolutionBcanbestoredinfreezerforupto4weekswhenkeptfromlightandmoisture.Avoidfreeze-thawcycles. - Determinetheoptimaldye/proteinratio(optional):
Note:Eachproteinrequiresdistinctdye/proteinratio,whichalsodependsonthepropertiesofdyes.Overlabelingofaproteincoulddetrimentallyaffectsitsbindingaffinitywhiletheproteinconjugatesoflowdye/proteinratiogivesreducedsensitivity.Werecommendyouexperimentallydeterminethebestdye/proteinratiobyrepeatingSteps4and5usingaserialdifferentamountoflabelingdyesolutions.Ingeneral4-6dyes/proteinarerecommendedformostofdye-proteinconjugates.- Use10:1molarratioofSolutionB(dye)/SolutionA(protein)asthestartingpoint:Add5µlofthedyestocksolution(SolutionB,assumingthedyestocksolutionis10mM)intothevialoftheproteinsolution(95µlofSolutionA)witheffectiveshaking.Theconcentrationoftheproteinis~0.05mMassumingtheproteinconcentrationis10mg/mLandthemolecularweightoftheproteinis~200KD.
Note:TheconcentrationoftheDMSOintheproteinsolutionshouldbe<> - Runconjugationreaction(seeStep4below).
- Repeat#3.2withthemolarratiosofSolutionB/SolutionAat5:1;15:1and20:1respectively.
- Purifythedesiredconjugatesusingpremadespincolumns.
- Calculatethedye/proteinratio(DOS)fortheabove4conjugates(seenextpage).
- Runyourfunctionaltestsoftheabove4conjugatestodeterminethebestdye/proteinratiotoscaleupyourlabelingreaction.
- Use10:1molarratioofSolutionB(dye)/SolutionA(protein)asthestartingpoint:Add5µlofthedyestocksolution(SolutionB,assumingthedyestocksolutionis10mM)intothevialoftheproteinsolution(95µlofSolutionA)witheffectiveshaking.Theconcentrationoftheproteinis~0.05mMassumingtheproteinconcentrationis10mg/mLandthemolecularweightoftheproteinis~200KD.
- Runconjugationreaction:
- Addtheappropriateamountofdyestocksolution(SolutionB)intothevialoftheproteinsolution(SolutionA)witheffectiveshaking.
Note:ThebestmolarratioofSolutionB/SolutionisdeterminedfromStep3.6.IfStep3isskipped,werecommendtouse10:1molarratioofSolutionB(dye)/SolutionA(protein). - Continuetorotateorshakethereactionmixtureatroomtemperaturefor30-60minutes.
- Addtheappropriateamountofdyestocksolution(SolutionB)intothevialoftheproteinsolution(SolutionA)witheffectiveshaking.
- Purifytheconjugation:
Thefollowingprotocolisanexampleofdye-proteinconjugatepurificationbyusingaSephadexG-25column.- PrepareSephadexG-25columnaccordingtothemanufactureinstruction.
- Loadthereactionmixture(directlyfromStep4)tothetopoftheSephadexG-25column.
- AddPBS(pH7.2-7.4)assoonasthesamplerunsjustbelowthetopresinsurface.
- AddmorePBS(pH7.2-7.4)tothedesiredsampletocompletethecolumnpurification.Combinethefractionsthatcontainthedesireddye-proteinconjugate.
Note1:Forimmediateuse,thedye-proteinconjugateneedbedilutedwithstainingbuffer,andaliquotedformultipleuses.Note2:Forlongertermstorage,dye-proteinconjugatesolutionneedbeconcentratedorfreezedried(seebelow).
References&Citations | CitationExplorer |
DeepSequencingAnalysisoftheEha-RegulatedTranscriptomeofEdwardsiellatardaFollowingAcidification
Authors:DGao,NLiu,YLi,YZhang,GLiu
Journal:Metabolomics(LosAngel)(2017):2153--0769
Suramininhibitscullin-RINGE3ubiquitinligases
Authors:KennethWu,RobertAChong,QingYu,JinBai,DonaldESpratt,KevinChing,ChanLee,HaibinMiao,IngerTappin,JerardHurwitz
Journal:ProceedingsoftheNationalAcademyofSciences(2016):E2011--E2018
GlycosaminoglycanmimicrybyCOAMreducesmelanomagrowththroughchemokineinductionandfunction
Authors:HelenePiccard,NeleBerghmans,EvaKorpos,ChrisDillen,IlseVanAelst,SandraLi,ErikMartens,SandraLiekens,SamNoppen,JoVanDamme
Journal:InternationalJournalofCancer(2012):E425--E436
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颜料是用着色的物质通常与被着色的物质混合在一起主要以无机化合物为主;染料是一种用来直接或经媒染剂作用而能附着在各种纤维和其他材料上的有色物质有的可以跟被染物质化合,多以有机化合物为主。 颜料不能上色,而染料能上色. 颜料和染料的区别 颜料是一种微细粉末状的有色物质,一般不溶于水、油和溶剂,但能均匀的分散在其中。颜料是色漆的次要成膜物质,在木材装饰过程中调制底漆、腻子以及木才着色,也经常使用颜料.
想请问一下,DAPI这个染料到底有没有膜通透性,我通过百度搜索查询关于DAPI染料的,基本上是说它能透过细胞膜对活细胞和死细胞均能染上蓝色;但是也有人说DAPI只可以透过死细胞膜,不能对活细胞进行染色,用以区分活死细胞,到底哪个是对的啊,蒙了!!!!!!
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