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GST Fusion Protein Purification from Yeast
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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)
Stock
Volume
Final
3.3M*Triethanolamine(pH7.2)
194µl*
40mM
0.5MEDTA(pH8)
80µl
2mM
5MNaCl
600µl
150mM
0.1MDTT
400µl
2mM
10mMAEBSF
0.4ml
0.2mM
1.5mg/mlleupeptin
200µl
15µg/ml
0.5mg/mlpepstatin
20µl
20µg/ml
1Mbenzamidine
20µl
1mM
0.5mg/mlaprotinin
400µl
10µg/ml
100mMb-glycerolphosphate
20µl
100µM
50mMNa-o-vanadate
200µl
0.5mM
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-Vanadate
200ml
0.5mM
100mMNa-pyrophosphate
2ml
10mM
2mg/mlPhosvitin
10µl
1µg/ml

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