PD-10-Desalting-Columns

GEHealthcareInstructions52-1308-00BBPD-10DesaltingColumnsPD-10DesaltingColumnscontains•30prepackeddisposablePD-10DesaltingColumnscontaining8.3mlofSephadex™G-25Medium•4adapters•InstructionsforusePurposePD-10DesaltingColumnsareprepackedanddesignedforrapid,convenientsampleclean-upofproteinsandotherlargebiomolecules(5000Mr).PD-10DesaltingColumnscanbeusedinawiderangeofapplicationssuchasdesalting,bufferexchangeandremovaloflow-molecularweightcompounds.p.2Tableofcontents1.Principle22.Adviceonhandling43.Safetyprecautions64.Columnassembly65.Gravityprotocol76.Spinprotocol87.Recoveryanddesaltingcapacity98.Columncharacteristics109.Orderinginformation111PrinciplePD-10DesaltingColumnscontainSephadexG-25Medium,whichallowsrapidgroupseparationofhighmolecularweightsubstancesfromlowmolecularweightsubstances.PD-10DesaltingColumnsareusedfordesalting,bufferexchangeandsamplecleanup.Smallmoleculeslikesalt,freelabelsandotherimpuritiesareefficientlyseparatedfromthehighmolecularweightsubstancesofinterest.Thechromatographytechniqueisgelfiltrationandmoleculesareseparatedonthebasisofdifferencesinsize.p.3•MoleculeslargerthanthelargestporesintheSephadexmatrixareexcludedfromthematrixandareelutedfirst,inorjustafterthevoidvolume.ThevoidvolumeisthecolumnvolumeoutsidetheSephadexmatrix.•MoleculessmallerthanthelargestporesintheSephadexmatrixwillpenetratetheporestovaryingextent.Theyhavealargeraccessiblecolumnvolumethanthelargemoleculesandthereforetheyeluteafterthelargemoleculesjustbeforeonetotalcolumnvolumeofbufferhaspassedthroughthecolumn.Groupseparationcanbemadeusingtwodifferentprotocols,gravityprotocolandspinprotocol.GEHealthcareprovidesanassortmentofsampleclean-upproducts.ThedifferentformatsavailablearesummarizedinTable1.Table1.Productoverview1Recommendedsamplevolumes.Clean-UpproductExclusionlimit,MrBedvolumeSamplevolumegravityprotocol1Samplevolumespinprotocol1PDSpinTrap™G-2550000.5ml-70to130μlPDMultiTrap™G-2550000.5ml-70to130μlPDMiniTrap™G-2550002.1ml0.1to0.5ml0.2to0.5mlPDMidiTrap™G-2550003.5ml0.5to1.0ml0.75to1.0mlPD-10DesaltingColumns50008.3ml1.0to2.5ml1.75to2.5mlPDMiniTrapG-107002.1ml0.1to0.3ml-PDMidiTrapG-107005.3ml0.4to1.0ml-p.42AdviceonhandlingProtocolselectionTheseparationcanbemadeusingtwodifferentprotocols,gravityprotocolorspinprotocol,seeTable2forprotocoloverview.GravityprotocolTheliquidpassesthroughthecolumnbygravityforce.•Thereisaslightlyhigherrecoveryanddesaltingcapacityusinggravityprotocolcomparedtowhenusingthespinprotocol.•Theappliedsampleisdiluted.SpinprotocolAdditionalgravityforceisaddedbyspinningthecolumninacentrifugeforsomeprotocolsteps.•Thereisnodilutionofthesample.Table2.Protocoloverview11.4timesdilutionvalidif2.5mlsamplevolumeisused.RecoveryTherecoveryofappliedamountsampleisdependentontypeofproteinorotherbiomolecule.Typicallytherecoveryisintherange70-90%.Anincreaseinsampleconcentrationcanimproverecovery.ProtocolSamplevolumeElutionbufferDilutionfactorDesaltingcapacityGravity1.0-2.5ml3.5ml1.4times198%Spin1.75-2.5mlNoneNone90%p.5Equilibration•ItiscriticaltoequilibratethecolumnsinceUVabsorbingstabilizersareusedincolumnpacking.•Equilibrationismostconvenientlymadebygravityalsowhenusingthespinprotocol.SampleApplication•ThePD-10Desaltingcolumnisintendedforsamplevolumesupto2.5ml.•Forsamplevolumeslessthan2.5ml,allowthesampletoenterthepackedbedcompletelyandthenaddequilibrationbuffer(stackervolume)sothatthetotalvolumeofsampleandbufferaddedequals2.5ml.•Allowthesampletoenterthepackedbedcompletelybeforeanyadditionofbufferforelution.LabMatebufferreservoirsTosimplifytheuseofPD-10columnsforgravityprotocoluseLabMate™bufferreservoirs.Fororderinginformation,seeSection9.•PlacetheLabMatebufferreservoirontopofthePD-10column.•Addallbuffer(25ml)thatshouldbeaddedinstep2intheGravityprocol(Section5)inonestep.Elution•Forhigherrecoveries,usestackervolumes(spinprotocol).Centrifugation•Forbetterresultifusingafixedanglerotorcentrifuge:Putthecolumnsinsamedirectioninallcentrifugationsteps(spinprotocol).p.63SafetyprecautionsAlwaysusenormalpersonalprotectiondeviceslikeglovesandsafetyglasseswhenhandlingPD-10DesaltingColumns.4ColumnassemblyForuseinthespinprotocolthePD-10DesaltingcolumnshouldbeassembledwithadapterandcollectiontubeasshowninFig1.Note:Thiscolumnassemblymayalsobeusedforconvenienthandlingofcolumnswhenusingthegravityprotocol.Fig1.Assemblyofcolumn,adapterandcollectiontube.WARNING:Thecolumnstoragesolution,0.15%Kathon™CG/ICPBiocide,ispotentiallyallergenic.Usegloveswhendiscardingthestoragesolution.p.75Gravityprotocol1PD-10Desaltingcolumnpreparation•Removethetopcapandpouroffthecolumnstoragesolution.•Cutthesealedendofthecolumnatnotch.2Columnequilibration•Fillupthecolumnwithequilibrationbufferandallowtheequilibrationbuffertoenterthepackedbedcompletely.•Repeat4times.•Discardtheflow-through.Note:About25mlequilibrationbuffershouldbeusedintotalforallthreesteps.3Sampleapplication•Addmaximum2.5mlofsampletothecolumn.•Forsamplevolumeslessthan2.5ml,addequilibrationbuffertoadjustthevolumeupto2.5mlafterthesamplehasenteredthepackedbedcompletely.•Letthesampleorequilibr