Mediomics/TR-FRET Bridge-It® L-Methionine (L-Met) Fluorescence Assay Kit, 384-well format/TRF-1-1-1006

TR-FRETBridge-It®L-Methionine(L-Met)FluorescenceAssayKit,384-wellformat 

Applications:

• DirectmeasureofL-Metlevelinsamples

• AssaysforenzymesinvolvedinusingMetasthesubstrateorMetastheproduct(i.e. methionineaminopeptidase)

Features:

• Easy: MixtestsampleorstandardwiththeAssaysolutionandincubate.Absolutelynowashsteps!

• Fast: Readfluorescentsignalafter50minutesofincubation

• Sensitive: AssaymeasuresL-Methionineatalowerlimitofdetection of10µM

• FlexIBLe: AssayisavailableinFRETandTR-FRETformat,canbeadaptedtocolorimetricformatuponrequest. 

• Specific:NocrossreactivitywiththestandardaminoacidsorL-Metderivatives(L-MetSulfoxideandL-MetSulfone).

BackgroundInformationonL-MethionineandtheTR-FRETBridge-It® FluorescenceAssay

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

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

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

Thecommonpropertyofallsequence-specificDNAbindingproteinsistheirABIlitytobindwithhighaffinityandspecificitytoaDNAduplexcontainingauniquenucleotidesequence-i.e.theDNAbindingsitefortheprotein.

Mediomics’novelassayplatformreliesonthiscommoncharacteristic.ADNAduplexcontainingthesequence-specificDNAbindingsiteforagiventargetDNA-bindingproteinissplitintotwoDNA“half-site”duplexeseachhavingashortsingle-strandedoverhang.Thesesingle-strandedextensionsareshortenoughsothatintheabsenceofthetargetproteinlittlespontaneousre-associationoccurs.Whenthetargetproteinispresent,however,itshighaffinityforthefull-lengthDNAsequencewilldrivethere-associationofthetwohalf-siteDNAduplexes.Thisre-associationcanbesensitivelydetectedbyincorporatingatwofluorophoresystem[adonor(Eu3+)andanacceptor]intoeachofthetwoDNAhalf-sites.Inthisassay,theDNAbindingproteinactsasabiosensorforitsspecificligand(SAM).TheligandbindstotheDNAbindingprotein,activatingthebindingsitebyaconformationalchange.ThepresenceoftheactivatedDNAbindingproteinisdetectedasanincreaseinfluorescencesignaloftheacceptorfluorophoreorasadecreaseinfluorescencesignalofthedonorfluorophoreasaresultofTimeResolvedfluorescenceresonanceenergytransfer(TR-FRET)betweenthetwofluorophores.

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: The100-wellkitisaone-timetrialpurchase.Forbulkpricingoptions,please contactus.Foranyotherquestionsorcomments,pleasedonothesitateto contactus.Wearealwayshappytohelp!

Forinformation ontheassayprinciplepleaseseethe TR-FRETBridge-It®AssayTechnologyPlatform pageordownloadthe protocol. Thisproductisprotectedbypatentsandpendingpatents.

TheTR-FRETversionoftheBridge-It®L-Methionine Assayisbrandnew,sotherearecurrentlynoreferencesfor it.However,belowarepublicationsthathaveusedourBridge-It®L-Methionine AssayKit.

SelectedCustomerPublicationsusingMediomics’Bridge-ItL-MethionineFluorescenceAssayKit:

1.BatraV,VermaP. DietaryL-methioninesupplementationmitigatesgamma-radiationinducedglobalDNAhypomethylation:EnhancedmetabolicfluxtowardsS-adenosyl-l-methionine(SAM)biosynthesisincreasesgenomicmethylationpotential. FoodChemToxicol.2014Jul;69:46-54.

—“…l-methionineandSAMassaykitwaspurchasedfromMediomicsInc.(St.Louis,MO,USA).”

2. MurrayTVA,DongX,SawyerGJ,CaldwellA,HalketJ,SherwoodR,QuagliaAetal.NADPHoxidase4regulateshomocysteinemetabolismandprotectsagainstacetaminophen-inducedliverdamageinmice.FreeRadicalBIOLOGyandMedicine.2015.

—“MethioninewasmeasuredinextractsfromWtandNox4-/-mouseliversusingaBridge-It®l-Methionine(l-Met)FluorescenceAssay(Mediomics)asperthemanufacturer’sprotocol.”

ClickhereforthefulllistofourCustomerPublications.

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#:TRF-1-1-1006A(100measurements-trialsize),TRF-1-1-1006B (384measurements)

Protocol: 

TR-FRETBridge-It®L-MethionineFluorescenceAssayKit,384-wellProtocol522.52KB

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MSDS: 

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

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SupportingProtocol: 

MeasurementofL-Metinbacterialcellandcellculture-384wellformat317.64KB

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