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当前位置: 首页 > 产品中心 > Other_kits > AAT Bioquest/Amplite™ Fluorimetric Beta-Galactosidase Assay Kit *Red Fluorescence*/12603/200 Tests
商品详细AAT Bioquest/Amplite™ Fluorimetric Beta-Galactosidase Assay Kit *Red Fluorescence*/12603/200 Tests
AAT Bioquest/Amplite™ Fluorimetric Beta-Galactosidase Assay Kit *Red Fluorescence*/12603/200 Tests
AAT Bioquest/Amplite™ Fluorimetric Beta-Galactosidase Assay Kit *Red Fluorescence*/12603/200 Tests
商品编号: 12603
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)571/585
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryCellBIOLOGy
ReporterGeneEnzymes
RelatedTagEnzymes
BiochemicalAssays
E.coli?-galactosidaseisa464kDtetramer.Eachunitof?-galactosidaseconsistsoffivedomains,thethirdofwhichistheactivesite.Itisanessentialenzymeincells.DeficienciesofthisenzymecanresultingalactosialidosisorMorquioBsyndrome.InE.coli,?-galactosidaseisproducedbytheactivationofLacZoperon.DetectionofLacZexpressionhasbecomeroutinetothepointofdetectionofasfewas5copiesof?-galactosidasepercell.ThiskitusesaredfluorogenicgalactosidasesubstratethatcansensitivelydistinguishLacZ+fromLacZ-cells.Thenon-fluorescentsubstategeneratesastronglyfluorescentproductuponreactionwithgalactosidase.ItcanbeusedeitherfordetectinggalactosidaseconjugatesinELISAtypeassaysystemsorformonitoringLacZgeneexpressionincells.Amplite™FluorimetricBeta-GalactosidaseAssayKitcomeswithalltheessentialcomponentswithanoptimizedassayprotocol.Itcanbeusedwithafluorescencemicroplatereader,afluorescencemicroscope,oraflowcytometer.Itmightalsobeusedforscreeninggalactosidaseinhibitorsorinducers.
SpectrumAdvancedSpectrumViewer

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Movemouseovergridtodisplaywavelength&intensityvalues.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

 1.PrepareassaymixforOne96-wellplate:

1.1   Prepareβ-galactosidasesubstratestocksolution(200X):Add50µLofDMSO(ComponentE)intothevialofResorufinβ-galactoside(ComponentA)tomakestocksolution(200X).

Note:25µLofβ-galsubstratesstocksolution(200X)isenoughforone96-wellplate.Unusedstocksolutionshouldbealiquotedandstoredat<-20oC.Keepfromlightandavoidrepeatedfreeze-and-thawcycles.

 

 

1.2   Prepare0.3%β-mercaptoethanolassaybuffer:Add30µLofb-mercaptoethanol(ComponentF)to10mLofReactionBuffer(ComponentB),andmixwell.

Note:Additionalbufferisneededforpreparingserialdilutionofβ-galactosidase,whichisusedtogenerateastandardcurve.

1.3   Prepareassaymixture:Add25µLofβ-galactosidasesubstratestocksolution(fromStep1.1)into5mLofassaybuffer(fromStep1.2)tomakeassaymixture.

Note1:Thisβ-galassaymixtureisenoughforone96-wellplate.Itisnotstable,useitpromptly.

Note2:Onecandivideunusedβ-galassaymixtureintosingleusealiquotsandstoredat-20oC.

 

2.Preparelysisbufferworkingsolution:

Makelysisbufferworkingsolutionbyadding5µLofβ-mercaptoethanol(ComponentF)to5mLofLysisBuffer(ComponentD)beforeuse.

Note:Alwaysadd0.1%β-mercaptoethanolintolysisbufferbeforelysingthecells.

3.Preparecellextractsfrommammaliancells:

3.1   TreatcellscontainingLacZgenewithtestcompoundsforadesiredperiodoftime.

3.2   Washthecellstwicewith1XPBS.Donotdislodgethecells.

3.3   Foradherentcells:Addlysisbufferworkingsolution(fromStep2)tothecultureplates.Recommendedvolumesforvariousplatesarelistedinthefollowingtable.

Typeofcultureplate

Volumeoflysisbufferworkingsolution(μL/well)

96-wellplate

50

24-wellplate

250

12-wellplate

500

6-wellplate

1000

60mmplate

2000

100mmplate

4000

 

Fornon-adherentcells:Pelletthecellsintocentrifugetube,andadd50-2000µL(dependingonthesizeofthecellpellet)of1Xlysisbuffertothetube.

3.4   Incubatethecellswithcelllysisbuffer(fromStep3.3)atroomtemperaturefor10-15minutes,andgentlyswirltheplatesortubesseveraltimestoensurecompletelysis.

3.5   Proceedtotheβ-galactosidaseassayorfreezethesampleat-80oCtilluse.

Note1:Agoodlysiscanalsobeobtainedbyaquickfreeze-and-thawcycle(freeze1-2hoursat-20oCto‑80oCandthawatroomtemperature).

Note2:Alternatively,centrifugethecelllysisfor2-3minutestopellettheinsolublematerial,andthenassaythesupernatant.

 

4.Runb-galactosidaseassay:

4.1   Thawthetubeorplateoflysedcellsatroomtemperatureifneeded.Performtheassaydirectlyonthe96-wellplateifthecellswereseededina96-wellplate.

4.2   Add50µL/wellofβ-galactosidasecontainingcellextracts(fromStep3.4)intoa96-wellsolidbackplate.

Note1:Ifnecessary,dilutethelysatein1XLysisBufferwhentransfectionefficiencyisveryhigh.Orreducethevolumeoflysisbufferwhentransfectionefficiencyislow.Ifthetransfectionisperformedina96-wellplate,orastablecelllinewasseededintoa96-wellplate,performtheassaydirectlyontheplate.

Note2:Forendogenousß-galactosidaseactivitycontrol,add50µLofcelllysatefromnon-transfectedcells.Forblankcontrol,add50µLoflysisbuffer.

4.3   Optional(ifastandardcurveisdesired):Prepareβ-galactosidase(E.Coli)standardsbyperforming1:3serialdilutionsin0.3%β-mercaptoethanolassaybuffer(fromStep1.2)togetapproximately100,30,10,3,1,0.3,0.1and0mU/mLβ-galactosidasestandards.Add50µL/wellofseriallydilutedb-galactosidasestandardsintoa96-wellsolidbackplate.

Note1:Adjustthestandardcurve(concentrationrange)tosuitthespecificexperimentalconditions,suchascelltype,number,transfectionefficiency,andsizeofthecultureplates.

 

 

Note2:Thedilutionsforthestandardcurvemustbepreparedfreshlyeachtimetheassayisperformed.

4.4   Add50µLofassaymixture(fromStep1.3)toeachwell.Incubatetheplateatroomtemperatureor37oCforapproximately10minutesto4hoursdependingonthecelltype.

4.5   Add50µLofStopBuffer(ComponentC)toeachwell.Thestopbuffercausesanincreaseinthefluorescenceintensityoftheproduct,inadditiontoterminatethereaction.

4.6   MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=540/590nm(cutoff570nm).

References&Citations
CitationExplorer

BZLF1AttenuatesTransmissionofInflammatoryParacrineSenescenceinEpstein-BarrVirus-InfectedCellsbyDownregulatingTumorNecrosisFactorAlpha
Authors:XubingLong,YuqingLi,MengtianYang,LuHuang,WeijieGong,ErshengKuang
Journal:JournalofVirology(2016):7880--7893


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。
品牌分类
淋巴细胞信号传导 GPCR抗体 羰基活性生物素 细胞代谢 胺反应生物素 链霉亲和素结合物 生物素化抗IgGs 乙酰基特异性抗体 细胞骨架抗体
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