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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Amplite™ Colorimetric Enterokinase Activity Assay Kit/11410/200 Tests
产品编号:11410
市  场 价:¥114560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$5728.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Amplite™ Colorimetric Enterokinase Activity Assay Kit/11410/200 Tests
商品介绍
Overview
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Ex/Em(nm)405/None
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryEnzymeDetection
ProteinKinases
RelatedPeptidasesandProteases
Enterokinase(alsocalledenteropeptidase)isaserineproteaseproducedbycellsintheduodenalwallandisakeyenzymeinhumanandanimaldigestionsystem.Enterokinaseconvertstrypsinogenintoitsactiveformtrypsin,resultinginthesubsequentactivationofpancreaticdigestiveenzymes.Thedeficiencyofenterokinaseresultsinintestinaldigestionimpairment.Theinhibitionofenterokinasemayhaveanti-tumoreffectsthroughsuppressingproteasesinvolvedincarcinogenesisandmetastasis.Therefore,highlyselectiveandsensitivedetectionofenterokinaseplaysakeyroleinbiochemicalapplications.Amplite™ColorimetricEnterokinaseActivityAssayKitoffersasensitiveassayforquantifyingenterokinaseactivity.Aftercleavageofenterokinase,theenterokinasesubstratecanbedetectedbyEKYellow™inanabsorbancemicroplatereaderat405nm.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
  1. Prepareseriallydilutedenterokinasestandardsandtestsamples:
    1. Add50µLofddH2O+0.1%BSAintoEnterokinaseStandardvial(ComponentD)tomake10µg/mLenterokinasestocksolution.
    2. Prepareenterokinasestandarddilutions:Add10µLof10µg/mLenterokinasestandard(fromstep1.1)into990µLofAssayBuffer(ComponentC)toget100ng/mLenterokinasesolution.Thenperform1:2serialdilutionsinassaybuffertogetapproximately50,25,12.5,6.25,3.13,and1.56ng/mLseriallydilutedenterokinasestandards.
    3. Addenterokinasecontainingsamplesandseriallydilutedenterokinasestandardsintoa96-wellclearbottommicroplateaccordingtoTables1and2.
  2. Table1.Layoutofenterokinasestandardsandtestsamplesina96-wellclearbottommicroplate
    BLBLTSTS......
    EK1EK1............
    EK2EK2
    EK3EK3
    EK4EK4
    EK5EK5
    EK6EK6
    EK7EK7
    Note:EK=EnterokinaseStandard,BL=BlankControl(assaybuffer),TS=TestSample.

    Table2.Reagentcompositionforeachwell
    EKStandardBlankControlTestSample
    SerialDilutions:50µLAssayBuffer:50µL50µL
    Note1:Addtheserialdilutionsofenterokinasestandardsfrom1.56µMto100µMintowellsfromEK1toEK7.
    Note2:TheEKstandardsareforpositivecontrolonly,andshouldnotbereliedonasaquantitationstandardforenzymeactivity

  3. PrepareEnterokinaseassaymixture:
    1. MakeEKYellow™stocksolution(100X):
      Add50µLofDMSO(ComponentE)intoEKYellow™(ComponentA)tomake100Xstocksolution.
    2. MakeEnterokinaseSubstratestocksolution(100X):
      Add50µLofDMSO(ComponentE)intoEnterokinaseSubstrate(ComponentB)tomake100Xstocksolution.
    3. Makeassaymixture:
      Add50µLofEKYellow™stocksolution(fromStep2.1)and50µLofEnterokinaseSubstratestocksolution(fromStep2.2)into5mLofAssayBuffer(ComponentC),andmixwelltomakeenterokinaseassaymixture(ComponentA+B+C).
      Note1:Theassaymixtureisenoughforone96-wellplate.Itisnotstable,useitpromptly.
      Note2:StoreunusedEKYellow™stocksolutionat-20ºC,avoidlightandrepeatedfreeze-thawcycles.
  4. Runenterokinaseassay:
    1. Add50µLofassaymixture(fromStep2.3)intoeachwellofenterokinasestandard,blankcontrol,andtestsamples(seeStep1.3)tomakethetotalassayvolumeof100µL/well.
      Note:Fora384-wellplate,add25µLofsample,25µLofassaymixtureintoeachwell.
    2. Incubatethereactionmixtureat37ºCfor30-60minutes.
    3. MonitortheabsorbanceincreasewithanabsorbanceplatereaderwithpathcheckonatODof405nm.
References&Citations
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  1. MelicherovaK,KrahulecJ,SafranekM,LiskovaV,HopkovaD,SzeliovaD,TurnaJ.(2016)OptimizationofthefermentationanddownstreamprocessesforhumanenterokinaseproductioninPichiapastoris.ApplMicrobiolBiotechnol.
  2. LiuY,RenL,GeL,CuiQ,CaoX,HouY,BaiF,BaiG.(2014)Astrategyforfusionexpressionandpreparationoffunctionalglucagon-likepeptide-1(GLP-1)analoguebyintroducinganenterokinasecleavagesite.BiotechnolLett,36,1675.
  3. MbanefoEC,KikuchiM,HuyNT,ShuaibuMN,CherifMS,YuC,WakaoM,SudaY,HirayamaK.(2014)CharacterizationofagenefamilyencodingSEA(sea-urchinspermprotein,enterokinaseandagrin)-domainproteinswithlectin-likeandheme-bindingpropertiesfromSchistosomajaponicum.PLoSNeglTropDis,8,e2644.
  4. SkalaW,GoettigP,BrandstetterH.(2013)Do-it-yourselfhistidine-taggedbovineenterokinase:ahandymemberoftheproteinengineer"stoolbox.JBiotechnol,168,421.
  5. ChenZ,HanS,CaoZ,WuY,ZhuoR,LiW.(2013)Fusionexpressionandpurificationoffourdisulfide-richpeptidesrevealsenterokinasesecondarycleavagesitesinanimaltoxins.Peptides,39,145.
  6. WangK,WangB,YangHL,PanL.(2013)Newstrategyforspecificactivationofrecombinantmicrobialpro-transglutaminasebyintroducinganenterokinasecleavagesite.BiotechnolLett,35,383.
  7. SantanaSD,PinaAS,RoqueAC.(2012)Immobilizationofenterokinaseonmagneticsupportsforthecleavageoffusionproteins.JBiotechnol,161,378.
  8. ChunH,JooK,LeeJ,ShinHC.(2011)DesignandefficientproductionofbovineenterokinaselightchainwithhigherspecificityinE.coli.BiotechnolLett,33,1227.
  9. YamashinaI.(2010)ThetrailofmystudiesonglycoproteinsfromenterokinasetotumorMarkers.ProcJpnAcadSerBPhysBiolSci,86,578.
  10. KubitzkiT,MinorD,MackfeldU,OldigesM,NollT,LutzS.(2009)Applicationofimmobilizedbovineenterokinaseinrepetitivefusionproteincleavagefortheproductionofmucin1.BiotechnolJ,4,1610.

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