| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/560 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | EnzymeDetection Phosphatases |
| Related | BiochemicalAssays |
1.Prepareassayreactionmixture:
MixthewholecontentofPhosphataseSubstrate(ComponentA)withReactionBuffer(ComponentB)andkeepfromlight.
2.Prepareseriallydilutedalkalinephosphatasestandards(0to100mU/mL):
2.1 Add100μLofdistilledH2Owith0.1%BSA(H2O-0.1%BSA)intothevialofalkalinephosphatasestandard(ComponentC,10units)togeneratea100units/mLalkalinephosphatasestandardsolution.
Note:Thealkalinephosphatasestandardsolutionisnotstable.Unusedsolutionshouldbealiquotedandstoredat-20oC.Avoidrepeatedfreezeandthawcycles.
2.2 Add10μLof100units/mLalkalinephosphatasestandardsolution(fromStep2.1)into990µLofH2O-0.1%BSAtogeneratea1,000mU/mLalkalinephosphatasestandardsolution.
2.3 Take100μLof1,000mU/mLalkalinephosphatasestandardsolution(fromStep2.2)toperform1:100andthen1:3serialdilutionstoget10,3,1,0.3,0.1,0.03,0.01,and0mU/mLseriallydilutedalkalinephosphatasestandards.
2.4 Addseriallydilutedalkalinephosphatasestandardsand/oralkalinephosphatasecontainingtestsamplesintoasolidwhite96-wellmicroplateasdescribedinTables1and2.
Note1:Preparecellsortissuesamplesasdesired.
Note2:Unusedserialdilutionsofalkalinephosphatasestandardshouldbediscarded.
Table1.Layoutofalkalinephosphatasestandardsandtestsamplesinasolidwhite96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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AS1 | AS1 | …. | …. | …. | …. |
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AS2 | AS2 |
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AS3 | AS3 |
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AS4 | AS4 |
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AS5 | AS5 |
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AS6 | AS6 |
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AS7 | AS7 |
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Note:AS=AlkalinePhosphataseStandards;BL=BlankControl;TS=TestSamples.
Table2.Reagentcompositionforeachwell
AlkalinePhosphataseStandards | BlankControl | TestSample |
SerialDilutions*:50μL | H2O-0.1%BSA:50μL | 50μL |
*Note:Addseriallydilutedalkalinephosphatasestandardsfrom10to0.01mU/mLintowellsfromAS1toAS7induplicate.
3.Runalkalinephosphataseassayinsupernatants:
3.1 Add50μLofassayreactionmixture(fromStep1)intoeachwellofalkalinephosphatasestandard,blankcontrol,andtestsamples(seeStep2.4,Table1)tomakethetotalalkalinephosphataseassayvolumeof100µL/well
Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
3.2 Incubatethereactionfor30to60minutesatroomtemperature,protectedfromlight.
3.3 Add50μLofAssayBuffer(ComponentD)intoeachwellofalkalinephosphatasestandard,blankcontrol,andtestsampleswithassayreactionmixture(seeStep3.2)tomakethetotalalkalinephosphataseassayvolumeof150µL/well
Note:Fora384-wellplate,add25μLofAssayBuffer(ComponentD)intoeachwell.
3.4 Incubatethereactionfor10to30minutesatroomtemperature,protectedfromlight.
3.5 Monitortheluminescenceincreasewithastandardluminescenceplatereader.
4.Runalkalinephosphataseassayincells:
4.1 Treatthecellsasdesired.
4.2 Removethegrowthmediumcompletelyfromthecellplate.
Note:Itisimportanttoremovethegrowthmediumcompletelyfromthecellplateduetotheinterferenceofthegrowthmediumwiththephosphatasesubstrate.
4.3 Make1:1dilutionofthe5mLassayreactionmixture(fromStep1)with5mLdistilledH2O.
4.4 Add100μL(96-wellplate)or50uL(384-wellplate)of1:1dilutedassayreactionmixture(fromStep4.3)intothecellwells(fromStep4.2).
4.5 Incubatethereactionatthedesiredtemperaturefor30to60minutes,protectedfromlight.
4.6 Add50μL(96-wellplate)or25uL(384-wellplate)ofAssayBuffer(ComponentD)intothecellwellscontainingassayreactionmixture(fromStep4.5).
4.7 Incubatethereactionatroomtemperaturefor10to30minutes,protectedfromlight.
4.8 Monitortheluminescenceincreasewithastandardluminescenceplatereader.
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Journal:Journalofendodontics(2012):1101--1105
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Authors:AlanMSmith,MonicaRJaime-Fonseca,LiamMGrover,SerafimBakalis
Journal:Journalofagriculturalandfoodchemistry(2010):4719--4724
Regulationoftheosteoblast-specifictranscriptionfactorOsterixbyNO66,aJumonjifamilyhistonedemethylase
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Journal:TheEMBOjournal(2010):68--79
AAT Bioquest常用产品报价单
| 产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
| AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |
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