PurificationSchemeforUbiquitinExpressionLysate

Step1:TaketheexpressionlysissupernatentandtreatwithconcentratedaceticaciduntilthepHdropstobetween4.5and5.0.Thesolutionwillturnmilkywhite,sincemanyofthebacterialproteinswillfalloutofsolution.Centrifugethesolutionat10,000RPMinaSorvallSS-34rotorfor10minutesat4^oC.ThenadjustthepHwithsodiumhydroxidesothatitis5.1.Thispreventsotherproteinsfromprecipitatingduringyourionexchangestep.Youcanalsosyringefilterthesupernatentina0.2micronfiltertoremoveotherprecipitateddebris.

Step2:Setupyourcationexchangecolumn.UsePharmaciaFastFlowSPresininaXK50orXK26stylecolumn.PleaserefertotheabovedatasheetsifyouarenotfamiliarwithionexchangeresinsortheoperationoftheXKcolumns.

Figure1.DiagramofXKstylecolumns.

Step3:Makeyourbuffers.AgoodbuffertouseforubiquitinpurificationatpH5.1isammoniumacetate,sincethebufferisvolatileandmassspectroscopycanbeperformeddirectlyonthefractions.Otherbufferoptionsareavailable(seereferencechartsandtablesbutdependonworkingpH).Twobuffersshouldbemade.BufferAisusuallythelowsaltbuffer(20mMammoniumacetate,pH5.1).BufferBisusuallythehighsaltbuffer(0.5Mammoniumacetate,pH5.1).WhenyouadjectthepH,besuretoalwaysuseeitherammoniumhydroxide(NH₄OH)oraceticacid,sincethegoalistonothaveanynon-volatilemetalionsinthesolutions.

Step4:Pre-equilibratethecationexchangecolumnbyfirstrunning2columnvolumesofhighsaltBufferB,followedbyextensivewashingby5columnvolumesoflowsaltBufferA.

Step5:Loadyourproteinsampleontothecolumn.Savethe"flowthrough"(thestuffthatdoesn"tsticktothecolumnjustincaseyouhadaproblem).Washthecolumnwith2to3columnvolumesofBufferA.ThenelutetheproteinfromthecolumnwithalineargrADIentofincreasingconcentrationofammoniumacetate(orBufferB).Collectthegradientin25mlfractions.

Step6:Analyzeyourfractionsinthemajorpeakofthegradient(determinedtypicallybyabsorbanceat280nm)byusingmassspectroscopyorgelelectrophoresis.Poolthepurefractionstogether,dialyzeagainstpurewater,andlyophilize(orfreeze-dry)theproteinsolutiontopowder.(AnHPLCstepusingaC18columnwithanacetonitrilegradientmaybenecessaryifthefractionsarenotsufficientlypure).