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AAT Bioquest/ReadiLink™ Protein Biotinylation Kit *Powered by ReadiView™ Biotin Visionization Technology*/5521/1
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | ProteinBiochemistry Generalproteins |
| Related | BiochemicalAssays |
Warmallthecomponentsbeforeopening,andimmediatelypreparetherequiredsolutionsbeforestartingtheconjugation.YoumightneedfurtheroptimizationforyourproteinlabelingsincethisSOPwasdevelopedforGoatanti-RabbitIgGlabeling.
1.Prepareproteinsolution(SolutionP):
Assumingtheconcentrationofthetargetproteinsolution(antibodysolution)is2mg/mL,mix5µL(10%ofthetotalreactionvolume)ofReactionBuffer(ComponentB)with50µLofthetargetproteinsolution.Ifyouhaveadifferenceproteinconcentration,adjusttheproteinvolumeaccordinglytomake~100µgproteinavailableforthislabelingreaction
Note1:ThepHoftheproteinsolution(SolutionP)shouldbe8.5±0.5.IfthepHoftheproteinsolutionislowerthan8.0,adjustthepHtotherangeof8.0-9.0usingReactionBuffer(ComponentB)orsaturatedsodiumbicarbonatesolution.
Note2:Theproteinshouldbedissolvedin1Xphosphatebufferedsaline(PBS),pH7.2-7.4.IftheproteinisdissolvedinTrisorglycinebuffer,itmustbedialyzedagainst1XPBS,pH7.2-7.4,toremovefreeaminesorammoniumsalts(suchasammoniumsulfateandammoniumacetate)thatarewidelyusedforproteinprecipitation.
Note3:ImpureantibodiesorantibodiesstABIlizedwithbovineserumalbumin(BSA)orgelatinmightnotbelabeledwell.Thepresenceofsodiumazideorthimerosalmightalsointerferewiththeconjugationreaction.Sodiumazideorthimerosalcanberemovedbydialysisorspincolumnforoptimallabelingresults.
Note4:Theconjugationefficiencyissignificantlyreducediftheproteinconcentrationislessthan2mg/mL.Foroptimallabelingefficiency,thefinalproteinconcentrationrangeof2-10mg/mLisrecommended.
2.PrepareReadiView™BiotinSEstocksolution(SolutionB):
Add10µLofDMSO(ComponentC)intothevialofReadiView™BiotinSE(ComponentA),andvortexthevialvigorously.
Note1:PreparetheReadiView™BiotinSEstocksolution(SolutionB)beforestartingtheconjugation.Usepromptly.ExtendedstorageoftheReadiView™BiotinSEstocksolutionmayreducethebiotinactivity.SolutionBcanbestoredinfreezerfortwoweekswhenkeptfrommoisture.
Note2:AliquottheReadiView™BiotinSEstocksolutioninto5vials(2µL/vial).ONLYonevialisneededforlabeling100ugproteins.Theremainingvialscanbestoredinfreezerfor2weeksincaseyouneedrepeatyourconjugation.
3.Runconjugationreaction:
3.1 Addtheproteinsolution(SolutionP)intothevialofReadiView™BiotinSEstocksolution(2µL/vial,SolutionB),andmixthemwellbyrepeatedlypipettingfor2-5minutes.
Note:TheDMSOconcentrationshouldbekeptlessthan10%.
3.2 Keeptheconjugationreactionmixtureatroomtemperaturefor30-60minutes.
Note:Theconjugationreactionmixturecanberotatedorshakenforlongertimeifdesired.
4.Preparespincolumnforsamplepurification:
4.1 InverttheSpinColumn(ComponentD)sharplyseveraltimestoresUSPendthesettledgelandremoveanybubbles.
4.2 SnapoffthetipandplacecolumninaWashingTube(2mL,ComponentE).Removethecaptoallowtheexcesspackingbuffertodrainbygravitytothesurfaceofthegelbed.Ifcolumndoesnotbegintoflow,pushcapbackintocolumnandremoveitagaintostarttheflow.Discardthedrainedbuffer,andthenplacethecolumnbackintotheWashingTube.
4.3 Centrifugefor1mininaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
4.4 Apply1-2mL1XPBS,pH7.2-7.4intothecolumn.AftereachapplicationofPBS,letthebufferdrainbygravity,orcentrifugethecolumnfor1mintoremovethebuffer.Discardbufferfromcollectiontube.Repeatfor3-4times.
4.5 Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
5.Purifytheconjugation:
5.1 Placethecolumn(fromStep4.5)inacleanCollectingTube(1.5mL,ComponentF).Carefullyloadthesample(20–100μL)directlytothecenterofthecolumn.
5.2 Afterloadingthesample,add1XPBS(pH7.2-7.4)tomakethetotalvolumeof110μL.Centrifugethecolumnfor5minutesat1,000xg,andcollectthesolutionthatcontainsthedesireddye-labeledprotein.
Note1:Forimmediateuse,thedye-proteinconjugateneedbedilutedwithstainingbuffer,andaliquotedformultipleuses.
Note2:Forlongertermstorage,thedye-proteinconjugatesolutionneedbeconcentratedorfreezedried(seebelow).
| References&Citations |  PrinterFriendlyVersion |
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