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-fusedproteinsarerenderedsolublewiththistechniquethoughenzymea

2、ctivitiycanrangefrom30-90%.MaterialsandReagents1.STEBuffer10mMTris-HCl,pH8.01mMEDTA150mMNaCl2.Lysozymesolution10mg/mlinwater(makefresh)3.PBS4.ElutionBuffer50mMTris.Cl,pH9.020mMGSH5.10%SarkosylinSTEBuffer6.10%TritonX-100inSTEBuffer7.1MDTT8.100mMIPTGPr

3、ocedure  Day11.Setupanovernightculturein50ml2XTYwith150mg/mlofampicillin. Day21.Seed5mlofovernightcultureto500ml2XTYwith150mg/mlofampicillin.2.Growat37oCtoanA600of0.6to0.8.3.Inducewith0.1mMto2mMofIPTG.Growfor3hrat37oCorgrowovernightatroomtemperature.

4、LowerIPTGconcentrationsandlowergrowingtemperaturestendtoproducegreatersolubilityattheexpenseofyield.4.Pelletcellsbycentrifugingat3000g,4oCfor10min.Decantmediaandresuspendcellsin30mlice-coldPBStowash.Transfertoa40-mlOakRidgetubeandcentrifugeat3000g,4o

5、Cfor10min.DecantPBS.5.Thisisaconvenientpointtostopandtostorepelletsat-80oC.Elsecontinuetolysecells.6.Thawpelletoniceifcellsarefrozenelseproceedtothenextstep.7.Resuspendpelletin10mloficecoldSTEBuffer.8.Add100mloffreshlypreparedlyozymesolution,incubate

6、onicefor15min.Justbeforesonication,add100mlof1MDTTand1.4mlof10%Sarkosyl.Mixthoroughlybyinversionandsonicateforatotaltimeof1min.9.Centrifuge16,000rpmfor20minontheSS34rotortopelletdebris.Transfersupernatanttoa50-mlconicaltubeanddiscardthepellet.Add4mlo

7、f10%TritonX-100andtopupwithSTEBufferto20ml.TheeffectiveconcentrationofSarkosylandTritonX-100willbe0.7%and2%respectively.Incubateatroomtemperaturefor30min.1.Pourthelysateto1mlbedofpreparedGlutathioneSepharoseinPBS.Incubateatroomtemperaturefor30minto1h

8、rwithagitation.Topreparethe50%slurry,shakeupthemediaandpipette2mltoa50mltube.Fillto50mlwithPBS,inverttubeafewtimes.Centrifugeto2000rpmonaswingbucketcentrifugethenswitchoff.CarefullysuckoffPBSandresuspendbeadswith1mlofPBS.2.Washthebeadswith3X50mlofPBS