gst融合蛋白纯化方法

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1、GST融合蛋白纯化方法 Purification of GST Fused ProteinsAbstract:ManypeoplehaveventedoutfrustrationoverinsolubleGST-fusedproteins.ThisisaprotocolforenzymaticallyactivesolubleGST-fusedproteins.AllGST-fusedproteinsarerenderedsolublewiththistechniquethoughenzymeactivitiycan

2、rangefrom30-90%.MaterialsandReagents1.STEBuffer10mMTris-HCl,pH8.01mMEDTA150mMNaCl2.Lysozymesolution10mg/mlinwater(makefresh)3.PBS4.ElutionBuffer50mMTris.Cl,pH9.020mMGSH5.10%SarkosylinSTEBuffer6.10%TritonX-100inSTEBuffer7.1MDTT8.100mMIPTGProcedure  Day11.Setupan

3、overnightculturein50ml2XTYwith150mg/mlofampicillin. Day21.Seed5mlofovernightcultureto500ml2XTYwith150mg/mlofampicillin.2.Growat37oCtoanA600of0.6to0.8.3.Inducewith0.1mMto2mMofIPTG.Growfor3hrat37oCorgrowovernightatroomtemperature.LowerIPTGconcentrationsandlowergr

4、owingtemperaturestendtoproducegreatersolubilityattheexpenseofyield.4.Pelletcellsbycentrifugingat3000g,4oCfor10min.Decantmediaandresuspendcellsin30mlice-coldPBStowash.Transfertoa40-mlOakRidgetubeandcentrifugeat3000g,4oCfor10min.DecantPBS.5.Thisisaconvenientpoint

5、tostopandtostorepelletsat-80oC.Elsecontinuetolysecells.6.Thawpelletoniceifcellsarefrozenelseproceedtothenextstep.7.Resuspendpelletin10mloficecoldSTEBuffer.8.Add100mloffreshlypreparedlyozymesolution,incubateonicefor15min.Justbeforesonication,add100mlof1MDTTand1.

6、4mlof10%Sarkosyl.Mixthoroughlybyinversionandsonicateforatotaltimeof1min.9.Centrifuge16,000rpmfor20minontheSS34rotortopelletdebris.Transfersupernatanttoa50-mlconicaltubeanddiscardthepellet.Add4mlof10%TritonX-100andtopupwithSTEBufferto20ml.Theeffectiveconcentrati

7、onofSarkosylandTritonX-100willbe0.7%and2%respectively.Incubateatroomtemperaturefor30min.1.Pourthelysateto1mlbedofpreparedGlutathioneSepharoseinPBS.Incubateatroomtemperaturefor30minto1hrwithagitation.Topreparethe50%slurry,shakeupthemediaandpipette2mltoa50mltube.

8、Fillto50mlwithPBS,inverttubeafewtimes.Centrifugeto2000rpmonaswingbucketcentrifugethenswitchoff.CarefullysuckoffPBSandresuspendbeadswith1mlofPBS.2.Washthebeadswith3X50mlofPBS