0.5MNaCl2.92gNaCl

0.01MTris0.121gTrisbase

pHto8.3andbringupto100ml

0.1MNaOAc0.82gNaOAc

0.5MNaCl2.92gNaCl

pHto4.0andbringupto100ml

•Poura5mlaffigelbluecolumn(biorad)andwashwith50mlPrewashtoprepthecolumn(whenfirstusedoriflastusedinmorethanaweek).

•Washwith50mlQ,followedby50mlRunningBuffer.

•Washwith50ml1.4MNaCl,ifeluateiscolored,thenre-equilibrate.

•Washwith50mlRunningBuffer.

•Load1mlserum,saveflow-through,elutewith2bedvolumesrunningbuffer(theserumalbuminshouldsticktothecolumnandtheIgshouldflowthrough).

•Oniceslowlyaddsaturatedammoniumsulfateto45%(550mlsample+450mlsaturatedammoniumsulfate).Tiltovernightat4°C.

•Pelletbyspinningat3,000rpmfor10minutesat4°C.

•ResUSPendthepelletin1ml1XPBSonice(don’tvortexoragitate)anddialyzeagainstPBS.

•ThepharmaciaHiTrap1mlcolumncanbind10micromolesofpeptideorproteinpermlofbedvolume.PreptheHiTrapcolumnbywashingwith10ml50%Isopropanol,25%Isopropanol,10%Isopropanol,and10ml1mMicecoldHCl.

•Resuspendthepeptideorproteinin1ml1XCouplingBufferandloadthecolumn.Holdatroomtemperaturefor1hour.

•Washwith5ml1XCouplingBufferandsavetheflowthrough.

•Washwith6mlBufferA,6mlBufferB,and6mlBufferA.Holdatroomtemperaturefor30minutes.

•Washwith6mlBufferB,6mlBufferA,and6mlBufferB.WashwithStorageBufferandholdat4°C.

•Topurifytheantibody,loadtheaffigelconcentrateontothecolumnandholdatroomtemperaturefor10minutes.

•Washwith50ml10mMTris7.5andthenwashwith50ml10mMTris7.5/500mMNaCl.Elutewith5ml100mMGlycinepH2.5into1ml1MTris8.0.

•Dialyzeandconcentratewithacentricon30.

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