Overview

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SampleLabelingProtocol

Note:ThislabelingprotocolwasdevelopedfortheconjugateofGoatanti-mouseIgGwithICG.Youmightneedfurtheroptimizationforyourparticularproteins.

1.Prepareproteinstocksolution(SolutionA):

Mix100µLofareactionbuffer(e.g.,1M sodiumcarbonatesolutionor1MphosphatebufferwithpH~9.0)with900µLofthetargetproteinsolution(e.g.antibody,proteinconcentration>2mg/mlifpossible)togive1mLproteinlabelingstocksolution.

Note1:ThepHoftheproteinsolution(SolutionA)shouldbe8.5±0.5.IfthepHoftheproteinsolutionislowerthan8.0,adjustthepHtotherangeof8.0-9.0using1M sodiumbicarbonatesolutionor1MpH9.0phosphatebuffer.

Note2:Theproteinshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4.IftheproteinisdissolvedinTrisorglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforproteinprecipitation.

Note3:ImpureantibodiesorantibodiesstABIlizedwithbovineserumalbumin(BSA)orgelatinwillnotbelabeledwell.Thepresenceofsodiumazideorthimerosalmightalsointerferewiththeconjugationreaction.Sodiumazideorthimerosalcanberemovedbydialysisorspincolumnforoptimallabelingresults.

Note4:Theconjugationefficiencyissignificantlyreducediftheproteinconcentrationislessthan2mg/mL.Foroptimallabelingefficiencythefinalproteinconcentrationrangeof2-10mg/mLisrecommended.

2.Preparedyestocksolution(SolutionB):

AddanhydrousDMSOintothevialofICGdyestomakea10-20mMstocksolution.Mixwellbypipettingorvortex.

Note:Preparethedyestocksolution(SolutionB)beforestartingtheconjugation.Usepromptly.Extendedstorageofthedyestocksolutionmayreducethedyeactivity.SolutionBcanbestoredinfreezerfortwoweekswhenkeptfromlightandmoisture.Avoidfreeze-thawcycles.

3.Determinetheoptimaldye/proteinratio(optional):

Note:Eachproteinrequiresdistinctdye/proteinratio,whichalsodependsonthepropertiesofdyes.Overlabelingofaproteincoulddetrimentallyaffectsitsbindingaffinitywhiletheproteinconjugatesoflowdye/proteinratiogivesreducedsensitivity.Werecommendyouexperimentallydeterminethebestdye/proteinratiobyrepeatingSteps4and5usingaserialdifferentamountoflabelingdyesolutions.Ingeneral4-6dyes/proteinarerecommendedformostofdye-proteinconjugates.

3.1   Use10:1molarratioofSolutionB(dye)/SolutionA(protein)asthestartingpoint: Add5µlofthedyestocksolution(SolutionB,assumingthedyestocksolutionis10mM)intothevialoftheproteinsolution(95µlofSolutionA)witheffectiveshaking.Theconcentrationoftheproteinis~0.05mMassumingtheproteinconcentrationis10mg/mLandthemolecularweightoftheproteinis~200KD. 

Note:TheconcentrationoftheDMSOintheproteinsolutionshouldbe<10%.

3.2   Runconjugationreaction(seeStep4below).

3.3   Repeat#3.2withthemolarratiosofSolutionB/SolutionAat5:1;15:1and20:1respectively.

3.4   Purifythedesiredconjugatesusingpremadespincolumns.

3.5   Calculatethedye/proteinratio(DOS)fortheabove4conjugates(seenextpage).

3.6   Runyourfunctionaltestsoftheabove4conjugatestodeterminethebestdye/proteinratiotoscaleupyourlabelingreaction.

4.Runconjugationreaction:

4.1Addtheappropriateamountofdyestocksolution(SolutionB)intothevialoftheproteinsolution(SolutionA)witheffectiveshaking.

Note:ThebestmolarratioofSolutionB/SolutionisdeterminedfromStep3.6.IfStep3isskipped,werecommendusing10:1molarratioofSolutionB(dye)/SolutionA(protein).

4.2Continuetorotateorshakethereactionmixtureatroomtemperaturefor30-60minutes.

5.Purifytheconjugation

Thefollowingprotocolisanexampleofdye-proteinconjugatepurificationbyusingaSephadexG-25column.

5.1   PrepareSephadexG-25columnaccordingtothemanufactureinstruction.

5.2   Loadthereactionmixture(directlyfromStep4)tothetopoftheSephadexG-25column.

5.3   AddPBS(pH7.2-7.4)assoonasthesamplerunsjustbelowthetopresinsurface.

5.4   AddmorePBS(pH7.2-7.4)tothedesiredsampletocompletethecolumnpurification.Combinethefractionsthatcontainthedesireddye-proteinconjugate.

Note1:Forimmediateuse,thedye-proteinconjugateneedbedilutedwithstainingbuffer,andaliquotedformultipleuses.

Note2:Forlongertermstorage,dye-proteinconjugatesolutionneedbeconcentratedorfreezedried(seebelow).

CharacterizetheDesiredDye-ProteinConjugate

TheDegreeofSubstitution(DOS)isthemostimportantfactorforcharacterizingdye-labeledprotein.ProteinsoflowerDOSusuallyhaveweakerfluorescenceintensity,butproteinsofhigherDOS(e.g.DOS>6)tendtohavereducedfluorescencetoo.TheoptimalDOSformostantibodiesisrecommendedbetween2and10dependingonthepropertiesofdyeandprotein.Foreffectivelabeling,thedegreeofsubstitutionshouldbecontrolledtohave4-10molesofICGtoonemoleofantibody.ThefollowingstepsareusedtodeterminetheDOSofICGlabeledproteins.

1.Measureabsorption:

               Tomeasuretheabsorptionspectrumofadye-proteinconjugate,itisrecommendedtokeepthesampleconcentrationintherangeof1-10µMdependingontheextinctioncoefficientofthedye.

2.ReadOD(absorbance)at280nmanddyemaximumabsorption(ƛ_max=785nmforICGdyes):

Formostspectrophotometers,thesample(fromthecolumnfractions)needbedilutedwithde-ionizedwatersothattheODvaluesareintherangeof0.1to0.9.TheO.D.(absorbance)at280nmisthemaximumabsorptionofproteinwhile785nmisthemaximumabsorptionofICGdyes.ToobtainaccurateDOS,makesurethattheconjugateisfreeofthenon-conjugateddye.

3.CalculateDOSusingthefollowingequations:

3.1   Calculateproteinconcentration

3.2   Calculate

3.3   CalculatethedegreeoflabelingDOS=[Dye]/[Protein]=[^DOD₇₈₅´^Pε₂₈₀]/[230000×(A₂₈₀-0.073A₇₈₅)]

[Dye]isthedyeconcentration,andcanbereadilycalculatedfromtheBee-LambertLaw:A=ε_dyeCL.[Protein]istheproteinconcentration.Thisvaluecanbeeitherestimatedbytheweight(addedtothereaction)iftheconjugationefficiencyishighenough(preferably>70%)ormoreaccuratelycalculatedbytheBeer-LambertLaw:A=ε_proteinCL.Forexample,IgGhastheεvaluetobe203,000cm^-1M^-1.^Pε₂₈₀proteinmolarextinctioncoefficientat280nm(e.g.themolarextinctioncoefficientofIgGis203,000

cm^-1M^-1).CF(dyeabsorptioncorrectionfactorat280nm)=OD₂₈₀/OD₇₅₀= 0.073forICG-Sulfo-OSu.230,000cm^-1M^-1isthemolarextinctioncoefficientofICG-Sulfo-OSu.

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