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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/PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Blue Fluorescence*/21611/200 Tests
产品编号:21611
市  场 价:¥56560.00
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:$2828.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/PhosphoWorks™ Fluorimetric Pyrophosphate Assay Kit *Blue Fluorescence*/21611/200 Tests
商品介绍
Overview
PrinterFriendlyVersion

Ex/Em(nm)316/456
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategorySmallMoleculeDetection
Anions
RelatedCellSignalingMolecules
BiochemicalAssays
Pyrophosphate(PPi)isproducedbyanumberofbiochemicalreactions,suchasATPhydrolysis,DNAandRNApolymerizations,cyclicAMPformationbytheenzymeadenylatecyclaseandtheenzymaticactivationoffattyacidstoformtheircoenzymeAesters.OurPhosphoWroks™PyrophosphateAssayKitprovidesthemostrobustspectrophotometricmethodformeasuringpyrophosphate.Thiskitusesourproprietaryfluorogenicpyrophosphatesensorthathasitsfluorescenceintensityproportionallydependentupontheconcentrationofpyrophosphate.Ourassayismucheasierandmorerobustthantheenzyme-couplingpyrophosphatemethodsthatrequireatleasttwoenzymesfortheirpyrophosphatedetections.Thekitprovidesalltheessentialcomponentsforassayingpyrophosphate.Thiskithasbeensuccessfullyusedinhighthroughputscreening(HTS).PleaseinquirespecialHTSbulkpackagediscountforthescreeningof>10,000assays.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Prepareassaysolution:

1.1   Thawallthefourcomponentsatroomtemperaturebeforeuse.

 

1.2   Prepare200XPPiSensorStockSolution:Add50µLofDMSO(ComponentD)intothevialofPPiSensor(ComponentB)tomake200XPPiSensorStockSolution.

Note:25µLofthePPiSensorStockSolutionisenoughforone96-wellplate.TheunusedPPiSensorStockSolutionshouldbedividedintosingle-usealiquots.Storeat-20oCandprotectfromlight.

 

1.3   PrepareAssaySolution:Add25µLof200XPPiSensorStockSolution(fromStep1.2)to5mLofAssayBuffer(ComponentA),andmixthemwell.

Note1:DuetothehighsensitivityofthisassaytoPPi,itisimportanttousePPi-freelabwareandreagents.

Note2:DTT>1mMwillincreasethebackground,MgCl2>2mMdecreasetheresponse.

 

2.Prepareseriallydilutedpyrophosphatestandardsandtestsamples:

2.1   Prepare1mMPyrophosphateStandardSolution:Add10μLof50mMPyrophosphateStandard(ComponentC)into490μLofAssayBuffer(ComponentA),orbufferofyourchoice(preferably50mMHepesbuffer,pH7)tomake1mMpyrophosphatestandardsolution.

 

2.2   Add50μLof1mMpyrophosphatestandardsolution(fromStep2.1)into450μLofAssayBuffer(ComponentA)toget100μMpyrophosphatestandardsolution,andthentake200μLof100μMpyrophosphatestandardsolutiontoperform1:3serialdilutionstoget30,10,3,1,0.3,0.1and0 μMseriallydilutedpyrophosphatestandards.

 

2.3   Addseriallydilutedpyrophosphatestandardsand/orpyrophosphate-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.

 

Table1Layoutofpyrophosphatestandardsandtestsamplesinasolidblack96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

PS1

PS1

….

….

….

….

 

 

 

 

 

 

PS2

PS2

 

 

 

 

 

 

 

 

 

 

PS3

PS3

 

 

 

 

 

 

 

 

 

 

PS4

PS4

 

 

 

 

 

 

 

 

 

 

PS5

PS5

 

 

 

 

 

 

 

 

 

 

PS6

PS6

 

 

 

 

 

 

 

 

 

 

PS7

PS7

 

 

 

 

 

 

 

 

 

 

Note:PS=PyrophosphateStandard,BL=BlankControl,TS=TestSample.

 

Table2Reagentcompositionforeachwell

PyrophosphateStandards

BlankControl

TestSample

SerialDilutions*:50μL

AssayBuffer:50μL

50μL

Note:*Addseriallydilutedpyrophosphatestandardsfrom0.3μMto100μMintowellsfromPS1toPS7.

 

3.Runpyrophosphateassay:

3.1   Add50μL/wellofAssaySolution(fromStep1.3)tothewellsofpyrophosphatestandards,blankcontrol,andtestsamples.Mixthereagentsthoroughly.

Note:Fora384-wellplate,add25μLofsampleand25μLofAssaySolutionintoeachwell.

 

3.2   Incubateatroomtemperaturefor10to30minutes.

References&Citations
CitationExplorer

BIOLOGicalstudiesandtarget-engagementofthe2-C-methyl-D-erythritol4-phosphatecytidylyltransferase(IspD)-targetingantimalarialagent(1R,3S)-MMV008138andanalogs
Authors:MaryamGhavami,EmilioFernandoMerino,ZhongKeYao,RubayetElahi,MorganSimpson,MariaFernandez-Murga,JoshuaHaydenButler,MichaelCasasanta,PriscillaKrai,MaximTotrov
Journal:ACSInfectiousDiseases(2017)

StructuralInsightsintoInhibitionofEscherichiacoliPenicillin-bindingProtein1B
Authors:DustinTKing,GregoryAWasney,MichaelNosella,AnitaFong,NatalieCJStrynadka
Journal:JournalofBiologicalChemistry(2017):979--993

StructuralInsightsintoInhibitionofEscherichiacoliPenicillin-bindingProtein1B
Authors:DustinTKing,GregoryAWasney,MichaelNosella,AnitaFong,NatalieCJStrynadka
Journal:JournalofBiologicalChemistry(2017):979--993

BioMarkersidentifiedbyurinarymetabonomicsfornoninvasivediagnosisofnutritionalrickets
Authors:MaoqingWang,XueYang,LihongRen,SongtaoLi,XuanHe,XiaoyanWu,TingtingLiu,LiqunLin,YingLi,ChanghaoSun
Journal:Journalofproteomeresearch(2014):4131--4142

Host-PathogenInteractionandSignalingMoleculeSecretionAreModifiedinthedpp3KnockoutMutantofCandidalusitaniae
Authors:AymanSabra,Jean-JacquesBessoule,VesselaAtanasova-Penichon,ThierryNoel,KarineDementhon
Journal:Infectionandimmunity(2014):413--422


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