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AAT Bioquest/ReadiLink™ Protein Biotinylation Kit/5520/2 Labelings
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | |
| Category | ProteinBiochemistry Generalproteins |
| Related | BiochemicalAssays |
Warmallthecomponentsbeforeopening,andimmediatelypreparetherequiredsolutionsbeforestartingtheconjugation.YoumightneedfurtheroptimizationforyourproteinlabelingsincethisSOPwasdevelopedforGoatanti-RabbitIgGlabeling.
1.Prepareproteinsolution(SolutionP):
Assumingtheconcentrationofthetargetproteinsolution(antibodysolution)is5mg/mL,mix20µL(10%ofthetotalreactionvolume)ofReactionBuffer(ComponentB)with200µLofthetargetproteinsolution.Ifyouhaveadifferenceproteinconcentration,adjusttheproteinvolumeaccordinglytomake~1mgproteinavailableforthislabelingreaction
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.Runconjugationreaction:
2.1 Addtheproteinsolution(SolutionP)intothevialofReadiLink™BiotinSE(ComponentA),andmixthemwellbyrepeatedlypipettingforafewtimesorvortexthevialforafewseconds.
2.2 Keeptheconjugationreactionmixtureatroomtemperaturefor30-60minutes.
Note:Theconjugationreactionmixturecanberotatedorshakenforlongertimeifdesired.
3.Preparespincolumnforsamplepurification:
3.1 InverttheSpinColumn(ComponentC)sharplyseveraltimestoresUSPendthesettledgelandremoveanybubbles.
3.2 TwistoffthebottomclosureoftheSpinColumn(ComponentC),andloosenthecap.Placethecolumnina12x75mmtesttube(notprovided).
3.3 Centrifugefor1mininaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
3.4 Apply1-2mL1XPBS(pH7.2-7.4)tothecolumn.AftereachapplicationofPBS,centrifugethecolumnfor2mintoremovethebuffer.Discardthebufferfromthe12x75mmtesttube.Repeatthisprocessfor3-4times.
3.5 Centrifugefor2minutesinaswingingbucketcentrifugeat1,000xg(seeCentrifugationNotessection)toremovethepackingbuffer.Discardthebuffer.
4.Purifytheconjugation:
5.1 Placethecolumn(fromStep3.5)inaclean12x75mmtesttube.Carefullyloadthesample(100–500μL)directlytothecenterofthecolumn.
5.2 Afterloadingthesample,add1XPBS(pH7.2-7.4)tomakethetotalvolumeof220μLto520μL.Centrifugethecolumnfor5minutesat1,000xg,andcollectthesolutionthatcontainsthedesiredbiotin-labeledprotein.
Note1:Forimmediateuse,thebiotin-proteinconjugateneedbedilutedwithdesiredbuffer,andaliquotedformultipleuses.
Note2:Forlongertermstorage,thebiotin-proteinconjugatesolutionneedbeconcentratedorfreezedried(seebelow).
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