GSTFusionProteinPurificationfromYeast

  • 5mlovernightcultureofyourfavoriteyeastinyourfavoritemedium.
  • Inoculate50mlandgrow30oCshakingO/NuntilOD600=0.8to1.2.ForSCDculturesuse1/500and1/1500dilutions.
  • Optional:addalpha-factorto2.5µM.Continueshakingat30oCfor60min.
  • Add1MNaN3to10mM(finalconcentration)andmoveculturestoice.Everythingmustremaincoldfromhereonout.
  • Spincells3K,10minat4oC.
  • DiscardsupeandresUSPendcellswith0.5ml10mMNaN3.Transferto1.5mlmicrofugetube(notautoclaved).
  • Spin3K,10minat4oC.Alternativelyspin8K,1minatroomtemperature.
  • Discardsupe.Optional:freezepelletat-80oC.
  • Resuspendin1mlofcold10mMNaN3.
  • MeasureOD600.Adjustvolumessothatthereareanequalnumberofcellsineachsample.AtotalOD600of30persampleisbest.
  • Washwith1mlofLysisBuffer.
  • Spin3K10minat4oCanddiscardsupe.
  • Resuspendin400ulLysisBuffer.
  • Addascoopofglassbeadstoa0.5mlPCRtube.TransfercelllysatetothePCRtube
  • Vortex1min,4X.Keepsamplescoldbetweenvortexing.
  • Pokeaholeinthebottomofthetubeandspincelllysateinanewmicrofugetube1.5Kor500Xg,10min.,4oC.
  • Transferliquidfrombottomtubeintoanewmicrofugetube.
  • Spinagain1.5K,10min,4oCandagaintransferliquidintoanewmicrofugetube.
  • AddTritonX-100to1.5%androckfor60minat4oC.
  • Spin(3K,10min,4oC)andtransferthesupetoanewmicrofugetube.
  • Remove30ulofliquidandadd30ul2XSDSPAGESamplebuffer.ThiswillreflectproteincontentbeforeGlutathionepurification.
  • Totheremainingliquid,add100ul40%slurryofGlutathionebeadsandmixat4oCfor2h(overnightisusuallyfine).Glutathionebeadsshouldbeprewashed3XwithPBSand1Xwithlysisbufferbeforeresuspendingasa40%slurryinlysisbuffer.
  • WashglutathionebeadsfivetimeswithPBS,1%TritonX-100,300mMNaClatRT.Spin2K,5min,atroomtemperature.Rocksamplefor5minbetweenwashes.Changetubesafterthefirstwashtoreducenonspecificbindingtothetubeitself.
  • ResuspendinSDS-PAGESamplebuffer.
    • Alternativelyelute3timeswith1-2columnvolumesof5-10mMreducedglutathione,50mMTrispH8,andmixwith6XSDS-PAGEsamplebufferbeforestrippingthebeadswithSDS-PAGEsamplebuffer.
  • Heatto100oCfor10min.Thenstoreat-20oC.ProteinisreadytoberunonSDS-PAGEGel.
    • LysisBuffer(20ml)
    StockVolumeFinal
    3.3M*Triethanolamine(pH7.2)194µl*40mM
    0.5MEDTA(pH8)80µl2mM
    5MNaCl600µl150mM
    0.1MDTT400µl2mM
    10mMAEBSF0.4ml0.2mM
    1.5mg/mlleupeptin200µl15µg/ml
    0.5mg/mlpepstatin20µl20µg/ml
    1Mbenzamidine20µl1mM
    0.5mg/mlaprotinin400µl10µg/ml
    100mMb-glycerolphosphate20µl100µM
    50mMNa-o-vanadate200µl0.5mM
    HOHto20mls

    NOTES

    ThankstoPaulDiBelloandJiyoungChafortheirrefinementsofthisprotocol.

    *Indicatesacorrectionfromanearilerversionoftheprotocol.

    1.ForlysisinthepresenseofGDPandGTPIuseafinalconcentrationof10uMGDPor20uMGTPgammaSandafinalconcentrationof3mMMgCl2inthelysisbuffer.

    2.Youcansubstituteproteaseinhibitorcocktail(SigmaP8215)forindividualproteaseinhibitors(AEBSF,leupeptin,pepstatin,benzamidine,aprotinin).3.Forlysistodeterminephosphorylation,youmaywishtoaddmorephosphataseinhibitors(inadditiontobeta-glycerolphosphateandNa-o-vanadate)tothelysisbuffer,oryoumaywishtoomitphosphataseinhibitorsaltogether:

    50mMNa-M-Vanadate200ml0.5mM
    100mMNa-pyrophosphate2ml10mM
    2mg/mlPhosvitin10µl1µg/ml