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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/Amplite™ Cholesterol Quantitation Kit/40006/200 Tests
产品编号:40006
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/Amplite™ Cholesterol Quantitation Kit/40006/200 Tests
商品介绍
Overview
PrinterFriendlyVersion

Ex/Em(nm)571/585
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategorySmallMoleculeDetection
DiagnosticMolecules
RelatedBiochemicalAssays
Cholesterolisrequiredtobuildandmaintaincellmembranes.Itmodulatesmembranefluidityovertherangeofphysiologicaltemperatures.Withincells,cholesterolistheprecursormoleculeinseveralbiochemicalpathways.CholesterolisalsoanimportantprecursormoleculeforthesynthesisofVitaminDandthesteroidhormones,includingtheadrenalglandhormonescortisolandaldosteroneaswellasthesexhormonesprogesterone,estrogens,togetherwithtestosteroneandtheirderivatives.ThisAmplite™CholesterolQuantitationAssayKitprovidesoneofthemostsensitivemethodsforquantifyingcholesterol.ThekitusesAmplite™Redtoquantifytheconcentrationofcholesterol,whichisrelatedtotheproductionofhydrogenperoxideinthecholesteroloxidase-mediatedenzymecouplingreactionsinthepresenceofcholesterol.Theamountofcholesterolisproportionaltotheconcentrationofhydrogenperoxideformedintheenzymecouplingreactioncycle.Inthepresenceofperoxidase,thefluorescenceintensityofAmplite™Redisproportionaltotheconcentrationofhydrogenperoxidethatisconvertedtotheconcentrationofcholesterol.TheassaycanbereADIlyreadwithafluorescencemicroplatereaderatEx/Em=~540/590nm.
SpectrumAdvancedSpectrumViewer

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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

….

….

 

 

 

 

 

 

CHL1

CHL1

….

….

….

….

 

 

 

 

 

 

CHL2

CHL2

 

 

 

 

 

 

 

 

 

 

CHL3

CHL3

 

 

 

 

 

 

 

 

 

 

CHL4

CHL4

 

 

 

 

 

 

 

 

 

 

CHL5

CHL5

 

 

 

 

 

 

 

 

 

 

CHL6

CHL6

 

 

 

 

 

 

 

 

 

 

CHL7

CHL7

 

 

 

 

 

 

 

 

 

 

               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
Authors:Hai-BoTang,Zhuan-LingLu,Xian-KaiWei,Tao-ZhenZhong,Yi-ZhiZhong,Ling-XuanOuyang,YangLuo,Xing-WeiXing,FangLiao,Ke-KePeng
Journal:ScientificReports(2016)

HypermethylationoftheTGF-βtarget,ABCA1isassociatedwithpoorprognosisinovariancancerpatients
Authors:TimHMHuang,JacquelineShay,Yi-HuiLai,KennethPNephew,Hung-ChengLai,MichaelWYChan,Rui-LanHuang,Tai-KuangChao,PearllySYan,Yen-LingLai
Journal:(2015)

Antigenicallyshieldeduniversalredbloodcellsbypolydopamine-basedcellsurfaceengineering
Authors:BenWang,GuangchuanWang,BinjieZhao,JiajunChen,XueyunZhang,RuikangTang
Journal:ChemicalScience(2014):3463--3468


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