SmallscaleHis-Tagfusionproteinpurificationunderdenaturativeconditions Introduction Highlevelsofexpressionofrecombinantproteinsinabacterialsystemcanleadtotheformationofinsolubleaggregates,usuallyknownasinclusionbodies(IB).6MGuanidine-HCl(GuHCl),8MUreaorotherstrongdenaturantscanbeusedtocompletelysolubilizedIB.SinceunderdenaturatingconditionstheHistagiscompletelyexposed,itwillfacilitatethebindingtoNicolumns.Formostbiochemicalstudies,proteinhavetoberenaturedandrefolded,andthiscanbedoneinthecolumnitselfbeforeelutionorafterelution. AliquotofCellPelletafterInduction Theideaistoaliquotcellsafterinduction,andkeepat-80ºCenoughcellpelletsamplesforoptimizationofsmallscalepurificationprocedureandfurtherscale-up.Onceyousetupthebestpurificationconditionsatlowscale,youcanscale-uptheprocedure. EquilibrationofNi-NTAagarose Place50ulbeads(100ulsuspension)ofNi-NTAagarosebeadsin1.5mlplastictube. Washwith2x1.5mlH2Oand2x1.5mlequilibrationbuffer(washing:mix,spin3min3500rpm,dischargesupernatant). Buffers Lysisbuffer:50mMNa2HPO4pH8.0,0.3MNaCl,1mMPMSF(orproteaseinhibitorcocktailforbacterialcells#P-8849fromSigma)andstrongdenaturantas6MGuanidine-HCl(GuHCl)or6to8MUreaOptionaladditivestothelysisbuffera)1mMPMSForproteaseinhibitorcocktail1:200(cocktailforbacterialcells#P-8849fromSigma)b)Dnase100U/mlor25-50ug/ml(SIGMADN-25).Incubate10min4°Cinthepresenceof10mMMgCl2 Equilibrationbuffer:6to8MUrea,50mMNa2HPO4pH8.0,0.5MNaCl Washingbuffer:6to8MUrea,50mMNa2HPO4pH8.0,0.5MNaCl Elutionbuffer:6to8MUrea,20mMTrispH7.5,100mMNaCl,andappropriateimidazoleconcentrations. LimitationsDonotexposedNimatricestoreducingagentsasDTTorDTE(youcanuseßMEupto20mM);chelatingagentsasEDTAandEGTA;NH4+buffersandaminoacidsasArg,Glu,GlyorHis. Procedure 1Resuspendpelletof10mlbacterialculture(or100mlbacterialcultureforverylowexpressionlevel)in1mllysisbuffer 2Preparelysisbuffercontainingurea6to8MorGuanidine-HCl6M(try8MofUreafirst,andifproteinissolubletiterdowninthenextexperimentstillureaconcentrationtillminimalureaisrequiredforproteinsolubilization) 3Sonicateonice3x20seconds(dependsofthesonicator) 4Spin15minmaxspeed4°C 5Transfersupernatantintocleantube:crudeextract(keep40µlforPAGE-SDS) 6Equilibrate50µlNilbeadswithequilibrationbuffer(seeEquilibrationofNi-NTAagarose) 7Addthecrudeextracttothebeadsandincubate4°C/1h(swirl) 8Spin3min3500rpm.Dichargeunboundmaterial(keep40µlforPAGE-SDS) 9Wash3x1mlwithwashbufferwithappropriateureaconcentration.Washing:mix,spin3min3500rpm,dischargesupernatant(keep40µlforPAGE-SDS) 10Wash2x1mlwithwashbuffer+10mMimidazole(keep40µlforPAGE-SDS) 11Elutewith2x100µlelutionbuffer+100mMimidazole(keep40µlforPAGE-SDS) 12Finalelution:2x100µl250mMimidazole(keep40µlforPAGE-SDS) 13RunonPAGE-SDSgel5µlofcrudeextractandunboundmaterial,and13µlofthewashandelutionfractions. Analysisofresults-Troubleshooting IfproteindoesnotbindtotheNi-NTAresin,checktheintegrityoftheHistagbywesternblotusinganti-polyhistidineantibodies,ordoN-terminalsequencinginthecaseofN-terminaltags.Trytoworkat4°Callthetimeusingproteaseinhibitorsduringlysis. IftargetproteinelutewithproteincontaminantstrythealternativeprotocolwithincreasingImidazolconcentration.OrreduceamountofNi-NTAresin.OrtryadditivesasßME,glycerol,detergentsormoreNaClinthewashing/elutionbuffers. Ifelutedproteinseemtobedegradedtrytoworkat4°Callthetimeanduseproteaseinhibitorsduringlysis. Iftheproteindoesnotelutefromthecolumn(proteinisattachedtothecolumn)usehigherImidazolconcentrations(upto1M). Alternativeprotocoliftargetproteinisnotpureenough Performparallelpurificationprocedureswhereyouinclude10,20,30,40or50mMimidazoleinthelysis,bindingandwashingbuffer. Elutedirectlywith3x100ulelutionbuffer+250mMImidazol. CheckelutedproteinsonPAGE-SDS.ExpectloweryieldsbuthigherpurificationbyincreasingtheImidazolconcentration
