M2-magnetic-beads

ANTI-FLAG®M2MagneticBeadsCatalogNumberM8823StorageTemperature–20°CTECHNICALBULLETINProductDescriptionANTI-FLAGM2MagneticBeadsarecomposedofthemurinederived,ANTI-FLAGM2monoclonalantibodyattachedtosuperparamagneticironimpregnated,4%agarosebeadswithanaveragediameterof50μm.TheM2antibodybindstofusionproteinscontainingtheFLAG®peptidesequence.1Additionally,theM2antibodyrecognizestheFLAGoctapeptidesequence(N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C)attheN-terminus,Met-N-terminus,orC-terminuslocationsofafusionproteininmammalianandbacterialextracts.TheANTI-FLAGM2MagneticBeadsareusefulfordetectionandcaptureoffusionproteinscontainingaFLAGpeptidesequencebycommonlyusedimmunoprecipitationprocedures.Themagneticpropertiesallowforveryrapidseparationofthebeadsfromasuspension,significantlyacceleratingmanipulations,suchasrepetitivewashingsorprocessingofmultiplesamplesperformedinmultiwellplates.Thisleadstofasterexperimentation,betterreproducibility,andmoreaccuratequantitationoftheproteinsofinterest.Bindingcapacity:0.6mgofFLAGfusionproteinper1mlofpackedmagneticbeads.Specificity:≥90%specificitytowardsFLAGfusionproteinsfrommammalianandbacterialcellextracts.ReagentTheANTI-FLAGM2MagneticBeadresinissuppliedasa50%suspensionin50%glycerolwith10mMsodiumphosphate,150mMsodiumchloride,pH7.4,and0.02%(w/v)sodiumazide(PBA/A).PrecautionsandDisclaimerThisproductisforR&Duseonly,notfordrug,household,orotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.EquipmentRequiredButNotProvidedMagneticSeparatorsfor:Microcentrifugetubes(CatalogNumberM1167)Tissuecultureflasks(CatalogNumberM1292)Centrifugetubes(CatalogNumberM1542)Magnetfor96-welltissuecultureplates(CatalogNumberSHM05)Magneticplateforstandardsizedwellplates,T-25throughT-75tissuecultureflasks,andupto5cmdishes(CatalogNumberSHM04)Donotuseamagneticstirringsystem.Thiswilldestroytheresinbeads.Storage/StabilityTheANTI-FLAGM2MagneticBeadsshiponweticeandstorageat-20°Cisrecommended.Theproductissuppliedina50%glycerolsolutionwithpreservativeandisstablefor2yearsat-20°C.Afteruse,theresinshouldbecleanedandstoredin50%glycerolwithTBSorPBSbuffercontainingpreservativetoprotecttheproduct.Freezingthemagneticbeadsintheabsenceof50%glycerolwillirreversiblydamagethebeadstructure.ProceduresNote:Itisrecommendedtheentiretechnicalbulletinbereadbeforeuse,especiallytheReagentCompatibilityTableattheendofthisbulletin.Therearemanydifferentproceduresforperformingsmall-scaleaffinitycaptureexperiments.Thefollowingproceduresarewrittenforasinglesample.Forbatch-wisepurification,100μloftheresinbeadsuspensionperreaction(~50μlofpackedgel)isrecommended.Foruseina96-wellplateformat,10μlofpackedgelisrecommendedperwell.Theamountofresinbeadcanbevarieddependingontheamountoftargetproteininthesampleandthetypeofmagneticseparatorutilized.2PartI.SamplePreparation1.AdjustthepHoftheproteinextracttobetweenpH7-8.Itisalsousefultoadjustthesaltconcentrationwithsodiumorpotassiumchlorideto≥0.15Mintheproteinextracttopreventalargeamountofnonspecificproteinbindingtotheresin.2.TheFLAGfusionproteinextractmustbeclarifiedtoremoveanyinsolublematerial.Alargeamountofinsolublematerialmayrequirecentrifugation(10,000-20,000×gfor15minutes)forremoval.Theproteinextractshouldalsobefilteredthrougha0.45or0.22μmfiltertoremoveanyremainingcelldebrisandparticulatesthatmayinterferewithproteinbinding.PartII.BindingProceduresForpurificationofFLAGfusionproteins,theresincanbeusedineitherabatchor96-wellplateformat.Forlargervolumesoflysate,thebatchformatisrecommendedtoquicklycapturethetargetproteinfromalargevolumeofextract.Ifasmallersampleisbeingpurified,theFLAGfusionproteincanbeimmuno-precipitated.A.AutomationFormatforANTI-FLAGM2MagneticBeads-Pleaseseeourwebsite()forapurificationprocedureforFLAGfusionproteinsusingtheKingFisher®automatedplatform.B.BatchFormatforAbsorptionofFLAGFusionProteinswithANTI-FLAGM2MagneticBeads-ThisprocedureprovidesaquickandefficientwaytopurifyFLAGfusionproteinsfromadilutesolution.Table1.BindingCapacityofBeadsPackedGelVolume(μl)BindingCapacity(μg)20~1250~30100~60200~120TheANTI-FLAGM2MagneticBeadsarestoredin50%glycerolwithbuffer.Theglycerolmustberemovedjustpriortouseandtheresinequilibratedwithbuffer(steps1–5).Theequilibrationcanbedoneatroomtemperatureorat2–8°C.Removeonlythevolumeofresinthatisnecessaryforthepurification(seeTable1).DonotallowtheresintoremaininTBS(50mMTrisHCl,150mMNaCl,pH7.4)bufferforextendedperiodsoftime(24hours)unlessanantimicrobialagent(e.g.,0.02%sodiumazide)isaddedtothebuffer.1.Thoroughlyresuspendtheresinbygentleinversion.MakesurethebottleofANTI-FLAGM2MagneticBeadsisauniformsuspension.Removeanappropriatevolumeforuse(seeTable1).2.Transferresintoanappropriatesizedtube.Placetubeintheappropriatemagneticseparatortocollectthebeads.Removeanddiscardstoragebuffer.3.Equilibratebeadsbyresuspendingwith5packedgelvolumesofTBS(50mMTrisHCl,150mMNaCl,pH7.4).Mixthoroughly.4.Placetubeintheappropriatemagneticseparatortocollectthebeads.RemoveanddiscardTBSbuffer.5.Repeatsteps3and4once.Allowasmallamoun