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AAT Bioquest/Amplite™ Cholesterol Quantitation Kit/40006/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 571/585 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | SmallMoleculeDetection DiagnosticMolecules |
| Related | BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1.PrepareAmplite™Redstocksolution(250X):
Add40µLofDMSO(ComponentE)intothevialofAmplite™Red(ComponentA).Thestocksolutionshouldbeusedpromptly.Anyremainingsolutionneedstobealiquotedandrefrozenat-20°C.
Note1:Avoidrepeatedfreeze-thawcycles.
Note2:TheAmplite™Redisunstableinthepresenceofthiolssuchasdithiothreitol(DTT)and2-mercaptoethanol.ThefinalconcentrationofDTTor2-mercaptoethanolinthereactionshouldbenohigherthan10μM.TheAmplite™RedisalsounstableathighpH(>8.5).ThereactionsshouldbeperformedatpH7–8.Theprovidedassaybuffer,pH7.4,isrecommended.
2.Prepareseriallydilutedcholesterolstandards(0to10µM):
2.1 Add10μLof2mMCholesterolStandard(ComponentD)into990µLofAssayBuffer(ComponentB)togenerate20µMcholesterolstandard.
2.2 Take300μLof20µMcholesterolstandardtoperform1:3serialdilutionstoget10,3,1,0.3,0.1,0.03,0.01,and0 µMseriallydilutedcholesterolstandards.
2.3 Addseriallydilutedcholesterolstandardsand/orcholesterolcontainingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2.
Note:Treatthesamplesasdesired.
Table1.Layoutofcholesterolstandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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CHL1 | CHL1 | …. | …. | …. | …. |
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CHL2 | CHL2 |
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CHL3 | CHL3 |
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CHL4 | CHL4 |
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CHL5 | CHL5 |
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CHL6 | CHL6 |
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CHL7 | CHL7 |
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Note:CHL=CholesterolStandards,BL=BlankControl,TS=TestSamples
Table2.Reagentcompositionforeachwell
CholesterolStandards | BlankControl | TestSample |
Serialdilutions*:50μL | AssayBuffer:50μL | 50μL |
*Note:Addtheseriallydilutedcholesterolstandardsfrom0to10µMintowellsfromCHL1toCHL7induplicate.
3.Prepareassayreactionmixture:
3.1 Add5mLofAssayBuffer(ComponentB)intothebottleofCholesterolEnzymeMix(ComponentC),andmixthemwell.
3.2 Add20µLofAmpliteRed™stocksolution(250X,fromStep1)intotheCholesterolEnzymeMixbottle(fromStep 3.1).
4.Runcholesterolassay:
4.1 Add50μLofassayreactionmixture(fromStep3.2)intoeachwellofcholesterolstandard,blankcontrol,andtestsamples(seeStep2.3,Table1)tomakethetotalcholesterolassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofassayreactionmixtureintoeachwell.
4.2 Incubatethereactionatroomtemperatureor37°Cfor30minutes,protectedfromlight.
4.3 MonitorthefluorescenceincreaseatEx/Em=530-570nm/590-600nm(optimalatEx/Em=540/590nm)usingafluorescenceplatereader.
Note:Thecontentsoftheplatecanalsobetransferredtoawhiteclearbottomplateandreadbyanabsorbancemicroplatereaderatthewavelengthof576±5nm.Theabsorptiondetectionhaslowersensitivitycomparedtothefluorescencereading.
| References&Citations |  CitationExplorer |
ViperininhibitsrABIesvirusreplicationviareducedcholesterolandsphingomyelinandisregulatedupstreambyTLR4
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Journal:ScientificReports(2016)
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Journal:(2015)
Antigenicallyshieldeduniversalredbloodcellsbypolydopamine-basedcellsurfaceengineering
Authors:BenWang,GuangchuanWang,BinjieZhao,JiajunChen,XueyunZhang,RuikangTang
Journal:ChemicalScience(2014):3463--3468
