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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 Phosphate Assay Kit *Red Fluorescence*/21660/125 Tests
产品编号:21660
市  场 价:¥85560.00
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:$4278.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/PhosphoWorks™ Fluorimetric Phosphate Assay Kit *Red Fluorescence*/21660/125 Tests
商品介绍
Overview
PrinterFriendlyVersion

Ex/Em(nm)571/585
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategorySmallMoleculeDetection
Anions
RelatedATPases
Phosphoproteins
Cellsutilizeawidevarietyofphosphate(Pi)andpolyphosphateestersasenzymesubstrates,secondmessengers,membranestructuralcomponentsandvitalenergyreservoirs.PhosphateisinvolvedinmanyBIOLOGicalprocesses.Forexample,phosphatases,ATPasesandseveralotherenzymescatalyzebiochemicalreactionsinwhichinorganicphosphateisreleasedfromaphosphoestersubstrate.Detectionofmanyphosphoester-metabolizingenzymesisdifficultbecausesuitablesubstratesarenotavailable.ItusuallyhasbeennecessarytodetermineinorganicphosphatereleaseusingtediouscolorimetricassaysorrADIoisotope-basedmethods.ThisPhosphoWorks™FluorimetricPhosphateAssayKithasbeendevelopedformeasuringtheactivityofanyPi-generatingenzymeusingourredfluorescentphosphatesensor.ThekitprovidessensitivedetectionofPi,analternativetohazardousradioactivemethodsandotherlesssensitivecolorimetricassays.ThemeasurementofPiisbasedonthechangeintheabsorbanceandfluorescenceofournewphosphatesensor.Ourkitprovidesalltheessentialreagentsincludingphosphatesensor,phosphatestandardsandassaybuffer.Theassayisshowntoquantitatephosphateinsolutionatconcentrationsatleastdownto0.1µM.Itcanbeusedtomeasurethekineticsofphosphatereleasefromphosphatases(suchasGTPasesandATPases)bycouplingthetwoenzymaticreactions.Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformatandeasilyadaptedtoautomationwithnoseparationstepsrequired.
SpectrumAdvancedSpectrumViewer



Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

Note:Toachievethebestresults,it’sstronglyrecommendedtousetheblackplates.

 

1.PreparePhosphateSensorworkingsolution:

1.1   Thawoneofeachkitcomponentatroomtemperaturebeforeuse.

 

1.2   Make125XPhosphateSensorstocksolution:Add20μLofDMSOintoPhosphateSensor(ComponentB)tomake125XPhosphateSensorstocksolution.

 

1.3   MakePhosphateSensorworkingsolution:Transfer20μLof125XPhosphateSensorstocksolution(fromStep1.2)into2.5mLofsterileH2O,andmixwell

Note1:AvoiddirectexposureofPhosphateSensor(ComponentB)tolight.

Note2:AliquotandstoretheunusedAssayBuffer(ComponentA)andPhosphateSensorstocksolution(fromStep1.2)at-20oC.Avoidrepeatedfreeze/thawcyclesandpotentialPicontamination.

Note3:DuetothehighsensitivityofthisassaytoPi,itisextremelyimportanttousePi-freelaboratorywareandreagents.

 

2.Prepareseriallydilutedphosphatestandardsandtestsamples:

2.1   Preparephosphatestandard:Add50μLof1mMKH2PO4(ComponentC)into950μLofdeionizedwaterorenzymereactionbuffertogeta50μMphosphatestandardsolution.Thentake200μLof50μMphosphatestandardsolutiontoperform1:2serialdilutionstoget25,12.5,6.25,3.125,1.56,and0.78μMseriallydilutedphosphatestandards.

2.2   Addphosphatecontainingsamplesandseriallydilutedphosphatestandardsintoasolidblack96-wellmicroplateaccordingtoTables1and2.

 

Table1Layoutofphosphatestandardsandtestsamplesinasolidblack96-wellmicroplate

BL

BL

TS

TS

….

….

 

 

 

 

 

 

PS1

PS1

….

….

….

….

 

 

 

 

 

 

PS2

PS2

 

 

 

 

 

 

 

 

 

 

PS3

PS3

 

 

 

 

 

 

 

 

 

 

PS4

PS4

 

 

 

 

 

 

 

 

 

 

PS5

PS5

 

 

 

 

 

 

 

 

 

 

PS6

PS6

 

 

 

 

 

 

 

 

 

 

PS7

PS7

 

 

 

 

 

 

 

 

 

 

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

Table2Reagentcompositionforeachwell

PhosphateStandards

BlankControl

TestSample

SerialDilutions*:40μL

H2OorBuffer:40μL

40μL

Note:*Addtheserialdilutionsofphosphatestandardfrom0.1μMto50μMintowellsfromPS1toPS7.



3.RunPhosphoWorksTMfluorimetricphosphateassay:

Warning:RunthephosphateassayatpH6.5to7.4.

3.1   Add40μLofAssayBuffer(ComponentA)and20µLofPhosphateSensorworkingsolution(fromStep1.3)intoeachwellofphosphatestandard,blankcontrol,andtestsamples(seeStep2.2)tomakethetotalphosphateassayvolumeof100µL/well.

Note:Fora384-wellplate,add20μLofsample,20μLofAssayBuffer(ComponentA)and10µLofPhosphateSensorworkingsolution(fromStep1.3)intoeachwell.

 

3.2   Incubatethereactionmixtureatroomtemperaturefor15minutesto1hour.

 

3.3   MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm.

References&Citations
CitationExplorer

Sacrificialcrystaltemplatingofhyaluronicacid-basedhydrogels
Authors:RichelleCThomas,PaulEChung,ShanPModi,JohnGHardy,ChristineESchmidt
Journal:EuropeanPolymerJournal(2016)

Osteogeniccellculturescannotutilizeexogenoussourcesofsyntheticpolyphosphateformineralization
Authors:MarianneBAriganello,SidneyOmelon,FABIoVariola,RimaMWazen,PierreMoffatt,AntonioNanci
Journal:Journalofcellularbiochemistry(2014):2089--2102


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