Description: TheSNAP-CapturePullDownResinisusedtoselectivelycaptureandimmobilizeaSNAP-tag
® fusionproteinfromsolutionusingagarosebeads.Thesebeadshaveahighlo
ADI ngcapacityforSNAP-tagfusionproteinsandshowverylownon-specificabsorptionofproteinsfromacomplexlysate,makingthemespeciallysuitableforpulldownapplications.TheSNAP-CapturePullDownResinispreparedbythecouplingofSNAP-tagsubstratebenzylguaninewithahighlycross-linkedagarose(4%)withexcellentphysicalcharacteristics.TwomlofSNAP-CapturePullDownResinissufficienttoperform25pulldownassaysusing80µlofresins
USP ensionperassay.
TheSNAP-tagproteinlabelingsystemenablesthespecific,covalentattachmentofvirtuallyanymoleculetoaproteinofinterest.TheSNAPtagisbasedonhumanO6-alkylguanine-DNAalkyltransferase(hAGT).SNAP-tagsubstratesarefluorophores,biotinorbeadsconjugatedtoguanineorchloropyrimidineleavinggroupsviaabenzyllinker.Inthelabelingreaction,thesubstitutedbenzylgroupofthesubstrateiscovalentlyattachedtotheSNAP-tag.
Therearetwostepstotheuseofthissystem:subcloningandexpressionoftheproteinofinterestasaSNAP-tagfusion,andcaptureandimmobilizationofthefusionproteinusingSNAP-CapturePullDownResin.
Figure1. SubstratestructureonSNAP-CapturePullDownResin Notes: StorageofPullDownResin:DonotfreezetheSNAP-CapturePullDownResin.Storetheunusedresinat4°C.Withproperstorage,resinshouldbestableforatleastTwoyears.CorrectstorageandhandlingofSNAP-tagfusionproteinsisessentialtomaintainreactivityoftheSNAP-tagpriortoimmobilization.SNAP-tagfusionproteinscanbepurifiedbeforeimmobilization,buttheimmobilizationreactionalsoworksinnon-purifiedproteinsolutionsincludingcelllysates.Add1mMDTTtobuffersusedforthestorageofSNAP-tagfusionproteins.Proteinsamplesshouldbestoredat-20°C,orat-80°Cforlong-termstorage.Handlingattemperaturesabove0°CshouldbeminimizedbythawingtheSNAP-tagproteinsamplesshortlybeforeuse,andkeepingthemoniceuntiljustbeforetheimmobilization.Ifaparticularfusionproteinrequiresbufferswithoutreducingagents,payparticularattentiontominimizeallhandlingstepsoftheproteinabove4°Cbeforethelabelingreaction.TheSNAP-tagitselfistolerantofawiderangeofbuffers.Therequirementsofyourfusionpartnershoulddictatetheselectionofthebuffer.Thefollowingstoragebuffercompositionisrecommended,especiallywhenfreezingproteinmaterial:pHbetween7.0and8.0,monovalentsalts(e.g.sodiumchloride)between50mMand250mM,andatleast1mMDTT.Non-ionicdetergentscanbeaddedifrequired,butionicdetergentsshouldbeavoidedbecausetheyreducetheactivityoftheSNAP-tag.Manyproteinsbenefitfromtheadditionofglycerolforfrozenstorage,typically20%v/v.ImmobilizedSNAP-tagfusionproteinsshouldbestoredat4°C.Sodiumazidemaybeaddedto2 mMfinalconcentrationtopreventbacterialgrowth.Dependingonthest
ABI lityofthefusionpartner,undertheseconditionstheimmobilizedproteinshouldbestableat2–6°Cforseveralmonths.TheSNAP-CapturePullDownResinshouldnotbefrozen.TheSNAP-tagfusionproteinislinkedtotheSNAP-CapturePullDownResinbyacovalentbond.Thereforetheimmobilizedproteinisessentiallyirrevers
IBL yboundtotheresin.ItisimportanthowevertopreservethefunctionalstabilityoftheproteinfusedtotheSNAP-tagasmuchaspossible.Werecommendhandlingtheimmobilizedfusionproteinandstoringbetweenuseat4°C,toprepareitjustbeforeuse,andtohandleitasgentlyaspossible.