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《gst融合蛋白純化方法》由會員上傳分享，免費在線閱讀，更多相關(guān)內(nèi)容在行業(yè)資料-天天文庫。
1、GST融合蛋白純化方法?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
