Overview

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Ex/Em(nm)	405/None
MW	N/A
CAS#	N/A
Solvent	N/A
Storage	F/D/L
Category	EnzymeDetection ProteinKinases
Related	PeptidasesandProteases

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.
Table1.Layoutofenterokinasestandardsandtestsamplesina96-wellclearbottommicroplate


BL	BL	TS	TS	...	...
EK1	EK1	...	...	...	...
EK2	EK2
EK3	EK3
EK4	EK4
EK5	EK5
EK6	EK6
EK7	EK7

Note:EK=EnterokinaseStandard,BL=BlankControl(assaybuffer),TS=TestSample.

Table2.Reagentcompositionforeachwell


EKStandard	BlankControl	TestSample
SerialDilutions:50µL	AssayBuffer:50µL	50µL

Note1:Addtheserialdilutionsofenterokinasestandardsfrom1.56µMto100µMintowellsfromEK1toEK7.
Note2:TheEKstandardsareforpositivecontrolonly,andshouldnotbereliedonasaquantitationstandardforenzymeactivity


2. 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.
3. 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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