罗氏蛋白酶抑制剂说明

DonoteatContentversion:January2013Storeat+2to+8°C1.WhatthisProductDoes1.1PropertiescOmplete,Mini,EDTA-freetabletsinhibitabroadspectrumofserineandcysteineproteases.IncontrasttoothercOmpletetabletstheydonotcontainEDTAleavingthestabilityandthefunctionofmetaldependantproteinsuneffected.TheaffinitypurificationofPoly-HistaggedfusionproteinsviaIMAC(immobilizedmetalaffinitychroma-tography)isalsofacilitated(nodialysisnecessary).Duetotheoptimizedcompositionofthetabletstheyshowexcellentinhibitionofserineandcysteineproteasesandarethereforeverywellsuitedfortheprotectionofproteinsisolatedfromanimaltis-sues,plants,yeastandbacteria.cOmplete,Mini,EDTA-freecontainsbothirreversibleandreversibleproteaseinhibitors.Metalloandasparticproteasesarenotinhibited.1.2Contents30individuallypackedcOmpleteProteaseInhibitorCocktailTabletsinfoilblisters.Eachtabletissufficientforavolumeof10mlsolution.1.3Stability�Thetabletsarestableat+2to+8°C,storeddry,untilexpirationdate.�Thestocksolutionisstablefor1–2weeks,storedat+2to+8°C,oratleastfor12weeksat15to25°C.1.4ApplicationUsedfortheinhibitionofserineandcysteineproteasesinbacterial,yeast,plantandanimalcellextracts.Fortheinhibitionofproteolyticactivityinsmallervolumes,inwhichEDTAmayinterferewithproteinstabilityorsubsequentassaysorpurificationmethods(e.g.,ImmobilizedMetalChelateAffinityChro-matography[IMAC]).2.HowtoUsethisProduct2.1HandlingCarefullypushthetabletthroughthefoilpackagingusingthebaseofyourthumb(andnotyoutfingernail)topreventthebreakageoftab-lets.Donoteatthetablets.2.2PreparationofWorkingSolutionsOnetabletcOmplete,Mini,EDTA-freeissufficientfortheinhibitionoftheproteolyticactivityin10mlextractionsolution.Ifveryhighproteo-lyticactivityispresent,onetabletshouldbeusedfor7mlextractionbuffer.Thetabletscanbeaddeddirectlytotheextractionmedium.Alternativelyastocksolution(7×conc.)canbeprepared.Ifitisnecessarytoinhibitproteolyticactivityinalargervolumewerec-ommendtousecOmpleteEDTA-free(1tabletfor50mlextractionsolution,seeOrderingInformation).ThecompositionofthecOm-pleteEDTAfreetabletisidenticaltothecOmplete,Mini,EDTA-freetablet,thereforecomparableresultsareachievedwithbothproducttypes.2.3Stocksolution(7×conc.)DissolveonetabletcOmplete,Mini,EDTA-freein1.5mlredist.H2Oorin100mMphosphatebuffer,pH7.0.3.ResultscOmplete,Mini,EDTA-freetabletsareappliedtostabilizethoseextractswherethestabilityoractivityofmetalcontainingproteinsshouldnotbeeffected.SinceEDTAinterfereswithIMAC(immobilizedmetalaffinitychromatography)cOmplete,Mini,EDTA-freeispreferen-ciallyusedintheisolationprocessofPoly-Histaggedfusionproteins.cOmpleteMini,EDTA-freetabletsinhibitefficientlyserineandcysteineproteasesinabroadrangebutnotmetalloproteases.Occasionally,asparticproteases(“acidproteases“)caninterfereuponisolationsfromanimaltissuesorbacterialextracts(1).Theseprote-ases,however,exhibitpronouncedactivitiesonlyatlowpHvalues.IfextractionorsingleisolationstepshavetobeperformedatthispHrangetheadditionofpepstatin*isrecommendedtoinhibitasparticproteaseactivityasaprecaution.TypicalvaluesfortheinhibitionofdifferentproteasesandproteasemixturesbycOmplete,Mini,EDTA-freeareshownintable1.Table1:InhibitionofdifferentproteasesbycOmpleteProteaseInhi-bitorTablets.OnecOmplete,EDTA-freetabletwasaddedper10mlincubationsolution.Pro-teolyticactivitywasdeterminedwiththeRocheAppliedScienceUniversalPro-teaseSubstrate(casein,resorufin-labeled*).Whenextractionsorsingle-stepisolationsarenecessaryintheacidpHrange,simplyincludepepstatin*alongwithcOmplete,EDTA-freetabletstoensureaspartic(acid)proteaseinhibition.Allexperimentswereperformedatroomtemperature.*availablefromRocheAppliedScienceProteaseresp.proteasemixtureEnzymeconcen-tration(mg/ml)pH-value%inhibitionafterimmediateadditiontotheprotease%inhibitionafter60minincubation(protease+cOmplete)at+15to+25°CPancreas-extract0.027.884%94%Thermolysin(Metalloprote-ase)0.00057.87%10%Chymotrypsin0.0020.027.87.890%60%100%98%Trypsin0.020.0027.87.870%81%74%86%Papain0.337.874%87%0113.04728688001➂RocheDiagnosticsGmbHRocheAppliedScience68298MannheimGermanyContactandSupportToaskquestions,solveproblems,suggestenhancementsorreportnewapplications,pleasevisitourOnlineTechnicalSupportSiteat: