Description: TheSNAP-CaptureMagneticBeadsareusedtoselectivelyimmobilizeandmagneticallyseparateaSNAP-tag
® fusionproteinfromsolutionusingmagneticagarosebeads.Thesebeadsshowalownon-specificabsorptionofproteinsfromacomplexlysate,makingthemsuitableforpulldownapplications.TheSNAP-CaptureMagneticBeadsarepreparedbythecouplingofSNAP-tagsubstratebenzylguaninewithhighlystable75-150µmsuperparamagneticparticles.TwomlofSNAP-CaptureMagneticBeadsissufficienttoperform25immobilizationassaysusing80µlofbeads
USP ensionperassay.
TheSNAP-tagisanoveltoolforproteinresearch,allowingthespecific,covalentattachmentofvirtuallyanymoleculeormodifiedsurfacetoaproteinofinterest.TheSNAP-tagisaproteinbasedonmammalianO
6 -alkylguanine-DNAalkyltransferase(AGT). SNAP-tagsubstratesarederivativesofbenzylpurinesandbenzylpyrimidines. Intheimmobilizationreaction,theSNAP-tagiscovalentlyattachedtothesubstitutedbenzylgroupoftheSNAP-CaptureMagneticBeads.
Therearetwostepstousingthissystem:sub-cloningandexpressionoftheproteinofinterestasaSNAP-tagfusion,andcaptureandimmobilizationofthefusionproteinusingSNAP-CaptureMagneticBeads.
Figure1:SubstratestructureonSNAP-CaptureMagneticBeads Notes: StorageofMagneticBeads:DonotfreezetheSNAP-CaptureMagneticBeads.Storetheunusedbeadsat4°C.Withproperstorage,beadsshouldbestableforatleasttwoyears.CorrectstorageandhandlingofSNAP-tagfusionproteinsisessentialtomaintainreactivityoftheSNAP-tagpriortoimmobilization.SNAP-tagfusionproteinscanbepurifiedbeforeimmobilization,buttheimmobilizationreactionalsoworksinnon-purifiedproteinsolutionsincludingcelllysates. Add1mMDTTtobuffersusedforthestorageofSNAP-tagfusionproteins.Proteinsamplesshouldbestoredat-20°C,orat-80°Cforlong-termstorage.Handlingattemperaturesabove0°CshouldbeminimizedbythawingtheSNAP-tagproteinsamplesshortlybeforeuse,andkeepingthemoniceuntiljustbeforetheimmobilization.Ifaparticularfusionproteinrequiresbufferswithoutreducingagents,minimizehandlingstepsoftheproteinabove4°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.Sodiumazidemaybeaddedto2mMfinalconcentrationtopreventbacterialgrowth.Dependingonthest
ABI lityofthefusionpartner,undertheseconditionstheimmobilizedproteinshouldbestableat2–6°Cforseveralmonths.TheSNAP-CaptureMagneticBeadsshouldnotbefrozen.TheSNAP-tagfusionproteinislinkedtotheSNAP-CaptureMagneticBeadsbyacovalentbond.Thereforetheimmobilizedproteinisessentiallyirrevers
IBL yboundtothebeads.ItisimportanthowevertopreservethefunctionalstabilityoftheproteinfusedtotheSNAP-tagasmuchaspossible.Werecommendhandlingtheimmobilizedfusionproteinandstoringbetweenuseat4°C,toprepareitjustbeforeuse,andtohandleitasgentlyaspossible.