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1、UniversalProteaseActivityAssay:CaseinasaSubstrateWrittenProtocol:A.AbstractProteasesbreakpeptidebonds.Inthelab,itisoftennecessarytomeasureand/orcomparetheactivityofproteases.Sigma'snon-specificproteaseactivityassaymaybeusedasastandardizedproceduretodeterminetheactivityofproteases,whichiswhatwedod
2、uringourqualitycontrolprocedures.Inthisassay,caseinactsasasubstrate.Whentheproteasewearetestingdigestscasein,theaminoacidtyrosineisliberatedalongwithotheraminoacidsandpeptidefragments.Folin&CiocalteusPhenol,orFolin’sreagentprimarilyreactswithfreetyrosinetoproduceabluecoloredchromophore,whichisqua
3、ntifiableandmeasuredasanabsorbancevalueonthespectrophotometer.Themoretyrosinethatisreleasedfromcasein,themorethechromophoresaregeneratedandthestrongertheactivityoftheprotease.Absorbancevaluesgeneratedbytheactivityoftheproteasearecomparedtoastandardcurve,whichisgeneratedbyreactingknownquantitiesof
4、tyrosinewiththeF-Creagenttocorrelatechangesinabsorbancewiththeamountoftyrosineinmicromoles.FromthestandardcurvetheactivityofproteasesamplescanbedeterminedintermsofUnits,whichistheamountinmicromolesoftyrosineequivalentsreleasedfromcaseinperminute.B.MaterialsReagents:Protease(P4630)PotassiumPhospha
5、te,Dibasic,Trihydrate(P5504)Casein(C7078)TrichloroaceticAcid(T0699)Folin&Ciocalteu’sPhenolReagent(F9252)SodiumCarbonate,Anhydrous(S2127)SodiumAcetate,Trihydrate(S8625)CalciumAcetate(C1000)L-Tyrosine,FreeBase(T3754)Equipment:0.45umpolyethersulfonesyringefilterandsyringe0.45µmDramvialsorpolypropyle
6、netubescapableofholding15mlsofsolutionSpectrophotometerCuvettesPipettesStir/HotplateStirbarScalepHMeterGraphingProgramC.PreparationofReagentsBeforebeginningtheassay,weneedtomakesurethatthefollowingreagentsarecorrectlyprepared:1.A50mMPotassiumPhosphateBuffer,pH7.5.Prepareusing11.4mg/mlofpotassiump
7、hosphatedibasic,trihydrateinpurifiedwaterandadjustingpHwith1MHCl.Thissolutionisplacedat37°Cpriortouse.2.A0.65%weight/volumecaseinsolution,preparedbymixing6.5mg/mlofthe50mMpotassiumphosphatebuffer.Thesolutiontemperature
