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当前位置: 首页 > 产品中心 > Other_kits > AAT Bioquest/PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit *UV absorption*/21659/200 Tests
商品详细AAT Bioquest/PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit *UV absorption*/21659/200 Tests
AAT Bioquest/PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit *UV absorption*/21659/200 Tests
AAT Bioquest/PhosphoWorks™ Colorimetric MESG Phosphate Assay Kit *UV absorption*/21659/200 Tests
商品编号: 21659
品牌: aatbio
市场价: ¥85560.00
美元价: 51336.00
产地: 美国(厂家直采)
公司:
产品分类: 其它检测试剂盒
公司分类: Other_kits
联系Q Q: 3392242852
电话号码: 4000-520-616
电子邮箱: info@ebiomall.com
商品介绍
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Ex/Em(nm)360/None
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategorySmallMoleculeDetection
Anions
RelatedATPases
Phosphoproteins
InthepresenceofinorganicphosphateMESGisconvertedto2-amino-6-mercapto-7-methlpurinebypurinenucleosidephosphorylase(EC2.4.2.1)withabsorptionwavelengthshifttored.ThisfeaturehasbeenusedtodevelopourconvenientMESGphosphateassaykit.OurkitprovidesalltheessentialreagentsincludingMESG,phosphorylaseandreactionbuffer.TheMESGsubstrategivesanabsorbanceincreaseat360nmonphosphorylysisatpH6.5-8.5,andatpH7.6thechangeinextinctioncoefficientis11,000M-1cm-1.Theassayisshowntoquantitatephosphateinsolutionatconcentrationsatleastdownto2µM.Itcanbeusedtomeasurethekineticsofphosphatereleasefromphosphatases(suchasGTPasesandATPases)bycouplingthetwoenzymaticreactions.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

1.Prepareassayreagents:

1.1   Thawallthefourcomponentsatroomtemperaturebeforeuse.

1.2   PrepareMESGSubstrate(ComponentB)Solution:Add500μLofddH2OtothevialofMESGSubstrate(ComponentB).MixwellbyvortexingtogetMESGSubstrateSolution.

Note:250ulisenoughforoneplatemakesingleusedaliquosandstoreitat-20°CImmediately.,

1.3   PreparePurineNucleosidePhosphorylase(ComponentC)Solution:Add100μLofddH2OtothevialofPurineNucleosidePhosphorylase(PNP;ComponentC).MixwellbyvortexingtogetPurineNucleosidePhosphorylaseSolution.

1.4   PrepareAssaySolution:AddthewholevolumeofMESGSubstrateSolution(fromStep1.2)andPurineNucleosidePhosphorylaseSolution(fromStep1.3)intothebottleofAssayBuffer(ComponentA),mixwelltogettheassaysolution.Placetheassaysolutiononice.

Note1:ThisAssaySolutionisstableforatleast4hoursonice.Itisnotrecommendedtofreezetheassaysolutionforanotherassay.

Note2:Toachievethedesirableresults,UV-transparentplatesorcuvettesarerequired.

Note3:DuetothehighsensitivityofthisassaytoPi,itisextremelyimportanttousePi-freelaboratorywareand

 

2.Prepareseriallydilutedphosphatestandardsand/ortestsamples:

2.1   PreparePhosphateStandard:Add50μLof1mMKH2PO4(ComponentD)into950μLofdeionizedwaterorenzymereactionbuffertogive50μMphosphatestandardsolution.

2.2   Take200μLof50μMphosphatestandardsolutiontoperform1:2serialdilutionstogive25,12.5,6.25,3.125,1.56,and0.78μMseriallydilutedphosphatestandards.

 

2.3   Addphosphate-containingtestsamplesand/orphosphatestandardsintoaclearUV-transparent96-wellmicroplateaccordingtoTables1and2.

 

Table1LayoutofphosphatestandardsandtestsamplesinaclearUV-transparent96-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

PhosphateStandard

BlankControl

TestSample

SerialDilutions*:50μL

Phosphate-freewaterorbuffer:50μL

50μL

*Note:Addtheserialdilutionsofphosphatestandardfrom0.1μMto50μMintowellsfromPS1toPS7.

3.RunPhosphoWorksMESGphosphateassay:

3.1   Add50μL/wellofAssaySolution(fromStep1.4)intothewellsofphosphatestandards,blankcontrol,andtestsamples.Mixthereagentsthoroughly.

Note:Fora384-wellplate,add25μLofsampleand25μLofAssaySolutionintoeachwell.

3.2   Incubateatroomtemperaturefor30minutes.Monitortheabsorbancewithamicroplatereaderorspectrophotometerat360nm.

Note:Forcuvetteassaythatrequiresthetotalvolumelargerthan100μL,multiplethevolumeofsampleandassayreagentproportionallybeforemeasuringtheabsorption.

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


品牌介绍
美国AAT Bioquest Inc.(前身是ABD Bioquest,Inc.)是一家为从事生命科学研究、诊断研发及药物开发的科学家研发、生产和销售生物分析研究试剂和试剂盒的公司。公司致力于光谱学检测领域,包括吸收(颜色)、荧光和发光技术。AAT Bioquest的产品帮助全世界的科学家和生物医药研究者更好的了解生物化学、免疫学、细胞生物学和分子生物学等领域。AAT Bioquest会不断研发新产品,快速地丰富各个领域的产品。 1)提供反应探针和发光探针,生物素和端粒酶能够应用于标记药物小分子和生物聚合物,如蛋白、核酸以及其他碳水化合物; 2)研究并生产荧光和发光探针用于检测蛋白,核酸和活细胞; 3)不断推出新型的荧光和发光探针用于检测多种酶,特别是检测水解酶和氧化还原酶类; 4)致力于开发用于信号转导研究的试剂; 5)提供生理和神经探针,特别是钙离子指示剂和膜电位探针。
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