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蚂蚁淘在线 / 品牌中心 / Everest Biotech / Everest Biotech/HRV 3C Protease 1 mg (1,000 Units)/1500010012/1 mg (1,000 Units)
商品描述
ProteinType:
Protease
Source:
E.coli
SpecificActivity:
>1Units/µg.1UnitsofTurbo3C(HRV3C)Proteasecleaves>95%of100µgoftargetproteinat4oCfor16hours.
Storage/Handling:
Storeat-80oC.
2mg/mlin50mMTris-HCl,pH8.0,150mMNaCl,1mMEDTA,1mMTCEP,50%glycerol
| A68kDaGST-fusionprotein(C)at1mg/mlisincubatedwithTurbo3C(HRV3C)Protease(*)ataratioof(1)1:50,(2)1:100,(3)1:200,(4)1:400(w/w)inabufferof25mMTris-HCl,pH8.0,150mMNaCl,14mMb-mercaptoethanolat4oCfor16hours.Thecleavedproductsare42kDaand26kDa |
CleavageinSolution
- MakefreshcoldDialysisBuffer.DialysisBuffershouldbeabufferinwhichthetargetproteinissoluble.ThereshouldbenoproteaseinhibitorintheDialysisBuffer.TheDialysisBuffershouldbecompatIBLewithdownstreampurificationprocesses,e.g.minimalamountofEDTAorDTTifNicolumnwillbeusedtoremovethecleavedHis-tag.
HereisanexampleofDialysisBuffer.25mMTris-HCl,pH8.0,150-500mMNaCl,14mMb-mercaptoethanol
Turbo3Chasthesameactivityin150mMNaClor500mMNaCland400mMimidazole.
- Dilutetheproteinpoolto1-2mg/mlwithDialysisBuffer.ThisisoptionalincasethetargetproteinaggregatesinDialysisBuffer.SaveasmallaliquotasUncutsampleforanalysis.EDTAmaybeaddedto0.5mMfinalconcentrationifthetargetproteinpooliselutedfromNicolumnandEDTAiscompatiblewiththetargetprotein.
- AddTurbo3CProteaseataProtease:targetproteinratioof1:100(w/w)or1,000unitTurbo3CProteaseto100mgoftargetprotein.Thereisnoneedtocalculatethemolarratio.Turbo3CProteasecanbeaddeddirectlytothetargetprotein.ThereisnoneedtochangebufferordiluteTurbo3CProtease.Theoptimalratioshouldbedeterminedempirically.AProtease-to-targetproteinratio(w/w)of1:50to1:200shouldworkformosttargetproteins.
- DialyzeagainsttheDialysisBufferat4oCovernight(about16hrs).DialysisistoremoveimidazoleorglutathioneifNiorglutathionecolumnisusedtoremovethecleavedtagorTurbo3CProteaseaftercleavage.Ifdesired,thetargetproteinpoolcanbebufferexchangedfirstbeforeRurbo3Ccleavage.
RemovalofTurbo3CProtease
- ThedialyzedtargetproteinandTurbo3CProteasemixturecanbeapplieddirectlytoaffinitycolumnsifcompatibleDialysisBufferisused.ForHis-taggedprotein,useIMACtoremovethecleavedHis-tagandTurbo3CProtease.ForGST-taggedprotein,useglutathionecolumntoremovethecleavedGST-tagandTurbo3CProtease.
- Ifdesired,analyzesamplesusingSDS-PAGEanalysis.ThedifferencebetweenthetaggedandcleavedtargetproteinmaybetoosmalltodetectbySDS-PAGE.ThecleavedHis-tagsometimescanbeseenatthebottomofthegel.
VishwanathaKBackN,MainsRE,EipperBA..AHistidine-richLinkerRegioninPeptidylglycineAlpha-AmidatingMonooxygenasehasthePropertiesofapH-sensor.JBiolChem.2014Mar13.KurutihalliVishwanatha,NilsBack,RichardE.MainsandBettyA.Eipper.Ahistidine-richlinkerregioninPeptidylglycinealpha-amidatingMonoocygenasehasthepropertiesofapH-sensor.TheJournalofBIOLOGicalChemistry,289,12404-12420.
Everest Biotech自2000年以来，我们一直是抗肽和抗原亲和纯化山羊多克隆抗体的专家。我们的抗体具有100％的满意保证-它们可在您的实验室或您的退款中发挥作用。 LifeSpan BioSciences，Inc.收购了Everest Biotech。 在此处查找使用Everest抗体的最新出版物盖玻片 用于悬浮细胞免疫荧光
