Mediomics/Bridge-It® L-Methionine (L-Met) Fluorescence Assay Kit, 96-well format/1-1-1005

Bridge-It®L-Methionine(L-Met)FluorescenceAssayKit,96-wellformat 

Applications:

• DirectmeasureofL-Metlevelinsamples

• AssaysforenzymesinvolvedinusingMetasthesubstrateorMetastheproduct(i.e. methionineaminopeptidase)

Features:

• Easy: MixtestsampleorstandardwiththeAssaysolutionandincubate

• Fast: Readfluorescentsignalafter50minutesofincubation

• Sensitive: AssaymeasuresL-Methionineatalowerlimitofdetection of10µM

• FlexIBLe: Assayisadaptabletobothlow-andhigh-throughputscreeningformats

 BackgroundInformationonL-MethionineandtheBridge-It® FluorescenceAssay

L-Methionine(L-Met)L-Methionineisanessentialaminoacidthatmustbeobtainedfromfoodsourcesorfromdietarysupplements.Itisaprecursorfortheothersulfuraminoacids,cysteine,taurine,andglutathione.MethioninereactswithadenosinetriphosphatetoformS-adenosylmethionine(SAM),whichistheprincipalmethyldonorinthebodyandcontributestothesynthesisofmanyimportantsubstances,includingepinephrineandcholine.L-methionineisalsoanimportantsupplementforanimalfeed.Currently,L-methionineisquantifiedusingthehighpressureliquidchromatography(HPLC)method.HPLCistimeconsuming,costly,and,duetothelargeamountoforganicsolventrequired,notenvironmentallyfriendly.

Bridge-It®L-MethionineFluorescence(L-Met)Assay^1,2

Eukaryoticcellscontainanestimated3,000sequence-specificDNAbindingproteins.Theseimportantproteins,actingeitherwithorwithoutaspecificsmallmoleculecoregulator(ligand),controlallaspectsofgenomicDNAactivityincludinggeneexpression,DNAreplication,andDNArepair.MediomicsisapplyingitsnovelfluorescenceassayplatformtodevelopinvitroassaysusefulforrapidlyandsensitivelyquantifyingtheactivityofbothDNAbindingproteinsandtheirsmallmoleculeligands.

TheMediomicsBridge-It®L-methioninefluorescenceassaymethodisbasedonacombinationofwell-establishedfluorescencemeasurementtechniquesandanewassayplatformdesignthatutilizesDNA-bindingproteinsasbiosensorsfortheirrespectivesmallmoleculecoregulators(ligands).TheaffinityoftheDNAsequence-specificMetJmethioninerepressorproteinforitsuniqueDNAbindingsiteisgreatlyincreasedinthepresenceofitsligand,S-adenosylmethionine(SAM).Forthisassay,theMetJconsensussequencewassplitintotwoapproximatelyequalDNA“half-sites”withonehalffragmentlabeledwithfluoresceinandtheotherhalffragmentlabeledwithOyster®645fluorophore^3,4.TherelativeamountofSAMpresentinatestsamplewillinfluencetheamountofDNA-MetJproteincomplexformationintheassay.Whenthiscomplexforms,itbringsthefluorescencelabeled-DNAhalf-sitesintocloseproximityandcausesameasurablechange(increase)influorescencesignalemissionthatcanbereADIlymeasuredusingamicroplatereader(wavelengthsettings:absorption485nm;emission665nm).TheL-methionineisreadilyconvertedtoSAMbyS-adenosylmethioninesynthetaseinthepresenceofATPandMg2+.L-MethionineconcentrationsintestsamplesaredeterminedusinganL-methioninestandardcurve.

TheBridge-It®L-methioninefluorescenceassaymethodexhibitshighlydesirableperformancecharacteristicsincludingahighsignaltobackground(S/B)ratio,agoodlineardynamicrange,and,alimitofdetectionof~10μM.Thetotalassaytimeis50minutes.

Note: Thisstandardcurveis forreferenceonly.Pleasegenerateyourownstandardcurve.

IncontrasttotheHPLCprocedure,theBridge-It®L-Methioninefluorescenceassaymethodutilizesthe384and96-wellmicroplateformat.Thus,itisideallysuitedfortherapid,simultaneousmeasurementofL-methioninelevelsinlargenumbersoftestsamples.Also,thismethodisreadilyadaptabletothehigh-throughputscreeningplatformscurrentlybeingusedindrugdiscoveryresearch.

Pricing: The50-wellkitisaone-timetrialpurchase.Forbulkpricingoptions,please contactus.Foranyotherquestionsorcomments,pleasedonothesitateto contactus.Wearealwayshappytohelp!

Forinformation ontheassayprinciplepleaseseethe Bridge-ItAssayTechnologyplatform pageordownloadthe protocol. Thisproductisprotectedbypatentsandpendingpatents.

SelectedCustomerPublicationsusing Mediomics’Bridge-ItL-MethionineFluorescenceAssayKit

1. Nicotinamidemetabolismregulatesglioblastomastemcellmaintenance.JungJ,KimLJY,WangX,WuQ,SanvoranartT,HubertCG,PragerBC,WallaceLC,JinX,MackSC,RichJN. JCIInsight.2017May18;2(10).pii:90019.

2. HepaticActivityandTranscriptionofBetaine-HomocysteineMethyltransferase,MethionineSynthase,andCystathionineSynthaseinPeriparturientDairyCowsAreAlteredtoDifferentExtentsbySupplyofMethionineandCholine.ZhouZ,GarrowTA,DongX,LuchiniDN,LoorJJ. JNutr.2017Jan;147(1):11-19.

3. NADPHoxidase4regulateshomocysteinemetabolismandprotectsagainstacetaminophen-inducedliverdamageinmice.MurrayTV,DongX,SawyerGJ,CaldwellA,HalketJ,SherwoodR,QuagliaA,DewT,AnilkumarN,BurrS,MistryRK,MartinD,SchröderK,BrandesRP,HughesRD,ShahAM,BrewerAC. FreeRadicBiolMed.2015Dec;89:918-30.

4. BatraV,VermaP.DietaryL-methioninesupplementationmitigatesgamma-radiationinducedglobalDNAhypomethylation:enhancedmetabolicfluxtowardsS-adenosyl-L-methionine(SAM)biosynthesisincreasesgenomicmethylationpotential.FoodChemToxicol.2014Jul;69:46-54.

1. Bridge-It®isaregisteredtrademarkofMediomics,LLC,St.Louis,Missouri,U.S.A.Mediomicshasaworldwide,exclusivelicenseforthisassayplatformfromSaintLouisUniversity,St.Louis,Missouri.

2. TheBridge-It®L-methioninefluorescenceassaykitisprovidedforlaboratoryR&Duseonly.ThisproductisnotapprovedbytheU.S.Governmentorbythegovernmentofanyothercountryoftheworldforuseinclinicaldiagnosisofdiseaseortreatmentofdiseaseinhumansoranimals.

3. Oyster®isaregisteredtrademarkofDenovoBiolabels,GmbH,Munster,Germany.

4. Flownamics®AnalyticalInstruments,Inc.,Madison,Wisconsin,istheauthorizedU.S.distributorofOyster®dyes.

Catalog#: 1-1-1005A(50measurements), 1-1-1005B (96measurements)

Protocol: 

Bridge-It®L-Methionine(L-Met)FluorescenceAssayKit,96-wellformat451.98KB

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MSDS: 

L-Methionine(L-Met)FluorescenceAssayKitSDS212.66KB

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SupportingProtocol: 

MeasurementofL-Metinbacterialcellandcellculture-96wellformat407.71KB

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