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蚂蚁淘在线 / 品牌中心 / Everest Biotech / Everest Biotech/TEV Protease (TurboTEV) 10 mg (100,000 Units)/1500020102/10 mg (100,000 Units)
商品描述
ProteinType:
Protease
Source:
E.coli
SpecificActivity:
>10Units/µg.1UnitofTurboTEVProteasecleaves>85%of3µgofcontrolsubstratein1hourat30oC.
Formulation
2mg/mlin25mMTris-HCl,pH8.0,50mMNaCl,1mMTCEP,50%glycerol
Storage/Handling:
Storeat-20oC
A49kDaGST-fusionprotein(C)at1mg/mlisincubatedwithTurboTEVorTEVProteaseataratioof(1)1:50,(2)1:100,(3)1:200(w/w)inabufferof25mMTris-HCl,pH8.0,150mMNaCl,14mMb-mercaptoethanolat4oCfor16hours.Thecleavedproductsare27kDaand22kDa."TEV"isacompetitors’TEVProteaseproduct.
Q)ForyourTEVdigestreaction,theprotocolstatesthatthereactionisperformedin25mMTris-HCl,pH8.0,150mMNaCland14mMβ-mercaptoethanol.IwaswonderinghowimportantthesebufferconditionsareandwhetherthepresenceofanyofthefollowingmayhaveanadverseaffectonthecleavageefficiencyofyourTurboTEV?
a)1mMDTT
b)10%Glycerol
c)20mMHistidine
d)500mMNaCl
e)100mMTris-HCl
f)Additionofβ-mercaptoethanol
A)
a)DTTof1mMshouldbefine;
b)10%Glycerolwouldnotberecommended;
c)20mMHistidinewouldnotberecommended;
d)500mMNaClwouldnotberecommended;
e)100mMTriswouldnotberecommended;
f)β-mercaptoethanolfunctionslikeDTT,soaddingβ-mercaptoethanolshouldnotbeaproblem.
Q)Dowehavetobufferexchangemyproteinpriortocleavage?
A)Itmaybenecessary.
Step1:CleavageinSolution
- MakefreshcoldDialysisBuffer
WhenmakingfreshcoldDialysisBuffer,pleasebeawarethat:
a)Thetargetproteinneedstobesolubleinthebuffer
b)ThebuffershouldNOTcontainproteaseinhibitor
c)TheDialysisBuffershouldbecompatIBLewithdownstreampurificationprocesses,e.g.minimalamountofEDTAorDTTifNicolumnwillbeusedtoremovethecleavedHis-tag.
HereisanexampleofDialysisBuffer.25mMTris-HCl,pH8.0,150-500mMNaCl,14mMb-mercaptoethanol.TurboTEVhasthesameactivityin150mMNaClor500mMNaCland400mMimidazole.
- Dilutethetargetproteinpool
i.Dilutethetargetproteinpoolto1-2mg/mlwithDialysisBuffer.
Note:ThisisoptionalincasethetargetproteinaggregatesinDialysisBuffer.
ii.SaveasmallaliquotasUncutsampleforanalysis.EDTAmaybeaddedto0.5mMfinalconcentrationifthetargetproteinpooliselutedfromNicolumnandEDTAiscompatiblewiththetargetprotein.
- AddTurboTEVProtease
i.AddTurboTEVProteaseataProtease:targetproteinratioof1:100(w/w)or10,000unit(1mg)TurboTEVProteaseto100mgoftargetprotein.
Note:
Thereisnoneedtocalculatethemolarratio.
TurboTEVProteasecanbeaddeddirectlytothetargetprotein.
ItisnotrequiredtochangebufferordiluteTurboTEVProtease.
Theoptimalratioshouldbedeterminedbytheuser;howeveraProtease-to-targetproteinratio(w/w)of1:50to1:200shouldworkformosttargetproteins.
- Dialysis
i.DialyzeagainsttheDialysisBufferat4oCovernight(about16hrs).ThisstepistoremoveimidazoleorglutathioneifNiorglutathionecolumnisusedtoremovethecleavedtagorTurboTEVProteaseaftercleavage.
Ifdesired,thetargetproteinpoolcanbebufferexchangedfirstbeforeTurboTEVcleavage.
Step2:RemovalofTurboTEVProtease
- Applytocolumns
ThedialyzedtargetproteinandTurboTEVProteasemixturecanbeapplieddirectlytoaffinitycolumnsifcompatibleDialysisBufferisused.
ForHis-taggedprotein,useIMACtoremovethecleavedHis-tagandTurboTEVProtease.
ForGST-taggedprotein,useglutathionecolumntoremovethecleavedGST-tagandTurboTEVProtease.
- Optional:SDS-PAGEanalysis
Ifdesired,analyzesamplesusingSDS-PAGEanalysis.ThedifferencebetweenthetaggedandcleavedtargetproteinmaybetoosmalltodetectbySDS-PAGE.ThecleavedHis-tagsometimescanbeseenatthebottomofthegel.
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