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Mostcurrent proteinpurificationmethods useagarosebeadscarryingaffinityfunctionalitiessuchasIMAC,Glutathione,orantibodies.Thechoiceofthesefunctionalgroupsdependsontheproteinofinteresttobepurified,andalargevarietyisavailable,includingpre-functionalizedbeadsthatcanbecoupledtobiomolecules(seeSEPMAG® proteinpurificationhandbook chapter4and5).
Agaroseismost frequentlyusedasproteinpurificationmatrix fortworeasons.First,agaroseshowsverylownon-specificbindingtoproteins,increasingthepurityoftheelutedproteinfraction.Secondly,agaroseparticlesasawholeconsistofahydrophilic,three-dimensionalmeshlargeenoughforproteinstodiffuseinto.Thismesh allowsbiomoleculestointeractwiththefirstmicrometers oftheoutershelloftheagaroseparticle,providingalargeinteractionsurfacebetweenthefunctionalgroupsandtheproteinsofinterest,therebyincreasingtheyieldsthatcanbeobtainedinproteinpurification.
Agarosematricesareavailable bothnon-magneticandwithamagnetite (Fe3O4)corethatrendersthemmagnetic.Non-magneticagarosebeadsare typicallylarger (40-150µmindiameter)thanmagneticbeadstoreduceback-pressureinchromatography.Magneticbeadsaretypicallysmaller(2-40µmindiameter).Smallerbeadsprovide moresurfaceareaandthereforehigherinteraction withtheproteinofinterest.However,forbestmagneticbehavioraminimalsizeof10µmshouldbeused,especiallywhenworkingwithferrimagneticbeads(seeSEPMAG® proteinpurificationhandbook chapter12).
Formaximumflexibility,thechemistryoftheaffinityfunctions,aswellasthesurface,shouldbe identicalbetweenmagneticandnon-magneticmaterials,allowingforeasyswitchingbetweenthetwosystems.
Themaindifferencebetweennon-magneticandmagneticagarosebeadsis thewaytheyareseparatedfromthesurroundingmedium.Inthefirstpurificationsteps,thismediumcanbebacterialorcelllysate,cellculturemediumoranykindofphysiologicalbuffer.Duringthewashandelutionstepsofproteinpurification,thismediumcontainsbuffersofdifferentproperties(e.g.saltconcentrations,pH)thatinducebindingofthespecificprotein,removalofcontaminants,andelutionoftheproteinofinterest.Viscosityofthesesolutionscanvary,e.g.whensubstanceslikeglycerolareaddedtoreducenon-specificbinding.Everyproteinpurificationprocessrequires severalseparationsteps,thereforeefficientseparationhasalargeimpactbothonprocessingtime,andpurificationsuccess.
Forallproteinpurificationexperiments,itisimportantto adjusttheamountofaffinitybeadstotheamountofproteininthestartingmaterial.Thisisimportantnotonlyforcostreasons:toolittleaffinitymaterialwillresultinincompletebindingoftheproteininthesolution.Toomanyaffinitybindingsitesoffered,ontheotherhand,willleadto bindingofotherproteins,makingthepurificationlessspecific,sothatadditionalpurificationstepsarerequiredinordertoobtainpureprotein.
Non-magneticagarosebeadscanbe separatedfromthemediumbycentrifugation,byfiltrationingravitycolumns,oronchromatographysystems.Switchingbetweenthesemethods(e.g.whenmovingtohigherpurificationscales)requiresoptimization.Whenproteinexpressionlevelsarelow, largeamountsofstartingmaterial needtobeappliedtorathersmallvolumesofagarosebeads,whichcanbeatime-consumingprocess.Forverysensitiveapplicationsrequiringonlymicrolitersofvolumes,suchasimmunoprecipitation,separationcouldbeincomplete,leADIngto eitherlossofmaterialorcontaminationwithagarosebeads.
IDAvs.NTA. Chelatingligandsnitrilotriaceticacid(NTA)andiminodiaceticacid(IDA)supportsimilarinteractionbetweenNi2+andimidazoleringsofapolyhistidinetag,butNTAcoordinatestheNi2+with4valenciesandIDAwithonly3(orangecircles).Thisdifferenceimpactsthequalityoftheresultingpurifiedproteinfraction.
Youwillfindthispostandmanymoreinterestingarticlesaboutusingbiomagneticseparationforproteinpurificationinour AdvancedGuidetoBiomagneticProteinPurification:
FREEDownload:TheAdvancedGuidetoBiomagneticProteinPurification
Withmagneticbeads,the principleofseparationstaysthesame regardlessofapplicationandvolumes.Testreactionsorsensitiveapplicationslikeimmunoprecipitation canbeperformedinfewmicroliters,andevenautomatedonmicroliterrobots.Purificationofproteinsfromlitersofmediumorcelllysatecanbedoneequallywell becausethehandlingvolumesarereducedsignificantly afterthefirstseparationstep
Author:Dr.RolandFabis,HeadofOperationsatCubeBiotechGmbH,Monheim,Germany
Dr.RolandFabisholdsaPhDininorganicchemistryfromtheUniversityofMuenster,Germany,whereheworkedonsilicon-organiccompounds,surfacemodificationwithsilanes,andapplicationsofthesesubstances.Dr.FabisjoinedtheQiagenheadquartersinHilden,Germanyin1995,whereheheldvariouspositionsinbasicresearch,productdevelopmentandtechnologytransfer,especiallyinthefieldsofmagneticparticlesynthesisandproteinpurification.Heisoneoftheco-foundersofCubeBiotechandresponsIBLeforthedevelopmentandproductionofproteinaffinitymaterials,includingmanufacturingofmagneticbeads.