PurificationofGST-fusionprotein

1)SpindowncellsandresUSPendinice-coldPBS(40mlper1Lofculture).LysecellsbyFrenchpress.Keepeverythingoniceunlessstatedotherwise.

2)Spintopelletcelldebris(12,000gfor10min.).Removesupernatantanddilutewithice-coldPBSto100ml.Add2mlof50%glutathionesepharose4Bslurry(seebelow)tosupernatant.Incubateatroomtemperaturefor30minutes.

3)Centrifugesuspensiontopelletsepharosematrix(500gfor5min.)andremovesupernatant(keepeverythingforanalysislater).

4)Washpelletwith10mlofPBS.Spintopelletandremovewash.Repeatwash2X.

5)Eluteproteinwith1mlofGEB.Incubateatroomtemperaturefor10min.Spintopelletsepharosematrix.Repeatelutionstep2X.

6)Add10-20unitsofthrombintotheeluate.Incubateatroomtemperaturefor2hours.Rungeltocheckthatdigestiscomplete(alsorunsamplefrompreviousstepstocheckthatthebindingandelutionofproteinissatisfactory).

7)Loadsampleontoagelfiltrationcolumn(previouslyequilibratedwith20mMphosphatebuffer)toseparateyourproteinfromtheGSTmoietyandthrombin.Rungeltocheck.Ifnecessary,repeatgelfiltrationstep(afterpoolingandconcentratingfractions).AlternativelytheGSTmayberemovebyglutathionesepharoseafterdialysisorgel-filtration.

8)PoolfractionsandconcentrateproteinforNMRanalysis.

PreparationofGlutathioneSepharose4B

1)Resuspendmatrixinbottlebygentleshaking.2)Remove2-4mlofsepharoseandpelletsepharosematrix(500gfor5min.)3)Decantsupernatantcarefullyandwashwith15-30mlofice-coldPBS(mixbyinversion).Spin500gfor5min.anddecantsupernatant.4)Add1.5-3mlofPBStogivea50%slurry.SeeTIBSarticle"PurificationofGSTfusionproteins"formorediscussion.

================  蚂蚁淘在线  ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。