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GenWay/HRV3C Protease/GWB-15AECB/1 mg (1000 units)
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4000-520-616
Description:
| ProductName | HRV3CProtease. |
|---|---|
| Description | Humanrhinovirus3Cprotease(HRV3CProtease)isacysteineproteasethatrecognizesthecleavagesiteofLeu-Glu-Val-Leu-Phe-Gln/Gly-Pro,commonlyreferredtoasthePreScissionSite.ItcleavesbetweenGlnandGly.HRV3CProteaseisarestrictiongraderecombinantformoftheHRV3Cproteasethathasrobustactivityat4°CwithhighspecificactivityandgreatstABIlity.Itdoesnotrequireanyspecialbufferforitsactivityandcanbeusedinabuffermostsuitableforthetargetprotein.HRV3CProteaseisa47kDaproteinwithbothGSTandHistagssoitcanbeeasilyremovedbyeitherNi-chelatingorGlutathione(GSH)resinalongwiththecleavedtag.TheactivityofHRV3CProteaseistestedusingacontroltargetprotein.One(1)μgofHRV3CProteasehasatleast1unitactivityconventionallyusedbyothersuppliers(unitofHRV3Cproteasecleaves>95%of100μgofcontroltargetproteinat4°Cfor16hours).Nonon-specificactivityhasbeenobservedunderthesameconditionwithHRV3CProteasetocontroltargetproteinratioof1:10.Prolongedincubation(severaldays)underthesameconditiondoesnotshowanynon-specificcleavage. |
| ProteinType | Protease. |
| Source | E.coli. |
| SpecificActivity | >1Units/mg.1UnitsofHRV3CProteasecleaves>95%of100mgoftargetproteinat4°Cfor16hours. |
| CleavageinSolution | 1.MakefreshcoldDialysisBuffer.DialysisBuffershouldbeabufferinwhichthetargetproteinissoluble.ThereshouldbenoproteaseinhibitorintheDialysisBuffer.TheDialysisBuffershouldbecompatIBLewithdownstreampurificationprocesses,e.g.minimalamountofEDTAorDTTifNicolumnwillbeusedtoremovethecleavedHis-tag. HereisanexampleofDialysisBuffer.25mMTris-HCl,pH8.0,150-500mMNaCl,14mMb-mercaptoethanol HRV3Chasthesameactivityin150mMNaClor500mMNaCland400mMimidazole. 2.Dilutetheproteinpoolto1-2mg/mlwithDialysisBuffer.ThisisoptionalincasethetargetproteinaggregatesinDialysisBuffer.SaveasmallaliquotasUncutsampleforanalysis.EDTAmaybeaddedto0.5mMfinalconcentrationifthetargetproteinpooliselutedfromNicolumnandEDTAiscompatiblewiththetargetprotein. 3.AddHRV3CProteaseataProtease:targetproteinratioof1:100(w/w)or1,000unitHRV3CProteaseto100mgoftargetprotein.Thereisnoneedtocalculatethemolarratio.HRV3CProteasecanbeaddeddirectlytothetargetprotein.ThereisnoneedtochangebufferordiluteHRV3CProtease.Theoptimalratioshouldbedeterminedempirically.AProtease-to-targetproteinratio(w/w)of1:50to1:200shouldworkformosttargetproteins. 4.DialyzeagainsttheDialysisBufferat4°Covernight(about16hrs).DialysisistoremoveimidazoleorglutathioneifNiorglutathionecolumnisusedtoremovethecleavedtagorHRV3CProteaseaftercleavage.Ifdesired,thetargetproteinpoolcanbebufferexchangedfirstbeforeRurbo3Ccleavage. |
| RemovalofHRV3CProtease | 1.ThedialyzedtargetproteinandHRV3CProteasemixturecanbeapplieddirectlytoaffinitycolumnsifcompatibleDialysisBufferisused.ForHis-taggedprotein,useIMACtoremovethecleavedHis-tagandHRV3CProtease.ForGST-taggedprotein,useglutathionecolumntoremovethecleavedGST-tagandHRV3CProtease. 2.Ifdesired,analyzesamplesusingSDS-PAGEanalysis.ThedifferencebetweenthetaggedandcleavedtargetproteinmaybetoosmalltodetectbySDS-PAGE.ThecleavedHis-tagsometimescanbeseenatthebottomofthegel. |
| Description | Humanrhinovirus3Cprotease(HRV3CProtease)isacysteineproteasethatrecognizesthecleavagesiteofLeu-Glu-Val-Leu-Phe-Gln-Gly-Pro,commonlyreferredtoasthePreScissionSite.ItcleavesbetweenGlnandGly.HRV3CproteaseisarestrictiongraderecombinantformoftheHRV3Cproteasethathasrobustactivityat4°Cwithhighspecificactivityandgreatstability.Itdoesnotrequireanyspecialbufferforitsactivityandcanbeusedinabuffermostsuitableforthetargetprotein.Turby3CProteaseisa47kDAproteinwithbothGSTandHistagssoitcanbeeasilyremovedbyeitherNi-chelatingorGluthathione(GSH)resinalongwiththecleavedtag. |
| ActivityandSpecificity | TheactivityofHRV3CProteaseistestedusingacontroltargetprotein.One(1)ugofHRV3CProteasehasatleast1unitactivityconventionallyusedbyothersuppliers(1unitofHRV3CProteasecleaves?95%of100ugofcontroltargetproteinforatleast4°Cfor16hours).Nonon-specificactivityhasbeenobservedunderthesameconditionwithHRV3CProteasetocontroltargetproteinratioof1:10.Prolongedincubation(severaldays)underthesameconditiondoesnotshowanynon-specificcleavage. |
| StorageandStability | StoreHRV3CProteaseat-20°C.HRV3CProteaseisstableatroomtemperatureforatleasttwoweekswithoutlossofanyactivity.Itretainsfullactivityafterincubationat37Cforoneweek. |
| CleavageCondition | ItisrecommendedtouseHRV3CProteaseataprotease-to-targetproteinratioof1:100(w.w)or1unitofHRV3CProteaseto100ugoftargetproteininabuffersuitableforthetargetproteinat4°Covernight,withthetargetproteinconcentrationat1-2mg/ml.Inmostcases,targetproteinsarecompletelycleavedwithaprotease-to-targetproteinratioof1:50to1:400or1unitofHRV3CProteaseto50-400ugoftargetprotein(asshowninFigure1).Theefficiencyofcleavagemayvaryduetothesequencesaroundthecleavagesite,theconformationandthesolubilityofthetargetprotein.Duetoitshighspecifity,moreHRV3CProtease(at1:10ratio)orlongercleavagetime(overaweekend)athighertemperature(37C)canbeusedtoachievehighcleavageefficiencywithoutnon-specificcleavageoftargetproteins. |
| RemovalofHRV3CProteaseafterCleavage | HRV3CProteasecontainsbothGSTandHIStags.Aftercleavageofthetargetprotein,HRV3CProteasecanbeeasilyremovedalongwiththetagsfromthecleavagereactionbyaffinitychromatographyonaNi-chelatingresinforHis-taggedtargetproteinorGSHresinforGST-taggedtargetprotein. |
AdditionalInformation:
| Name | HRV3CProtease |
|---|---|
| RelatedProductNames | Purification,CleavageofProteinHisandGSTTag;HRV3CProteaseCleaveTaggedProteins;PreScissionSiteSpecificTagsRemovalHRV3CProtease |
| Concentration | LotSpecific |
| Format | ThisHRV3CProteaseisinastoragebufferof25mMTris-HCL,pH8.0,50mMNaCl,1mMTCEP,and50%glycerol.ThestoragebufferiscompatiblewithNi-chelatingresinandGSHresinforon-columndigestion. |
| Storage | StoreHRV3CProteaseat-20C.HRV3CProteaseisstableatroomtemperatureforatleasttwoweekswithoutlossofanyactivity.Itretainsfullactivityinstoragebufferafterincubationat37Cforoneweek. |
| IntendedUse | ResearchUseOnly |
