Deprecated: Required parameter $cat_id follows optional parameter $type in /www/wwwroot/ebimall.com/systems/hong.php on line 2088

Deprecated: Required parameter $where follows optional parameter $tree_id in /www/wwwroot/ebimall.com/systems/hlb.php on line 3505
AntibodiesOnline/HDL and LDL/VLDL Cholesterol Assay Kit/ABIN2345055/192 tests188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
您好,欢迎您进入188进口试剂采购网网站! 服务热线:4000-520-616
蚂蚁淘商城 | 现货促销 | 科研狗 | 生物在线

AntibodiesOnline/HDL and LDL/VLDL Cholesterol Assay Kit/ABIN2345055/192 tests
Application
BiochemicalAssay(BCA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeHDLandLDL/VLDLCholesterolAssayKitmeasurestheHDLandLDL/VLDLcholesterollevelswithinserumorplasmasamples.ThekitprovidesreagentsforseparatingandquantifyingHDLandLDL/VLDLcholesterol.Theassayisbasedontheenzymedrivenreactionthatquantifiesbothcholesterolestersandfreecholesterol.Cholesterolestersarehydrolyzedviacholesterolesteraseintocholesterol,whichisthenoxidizedbycholesteroloxidaseintotheketonecholest-4-en-3-oneandhydrogenperoxide.Thehydrogenperoxideisthendetectedwithahighlyspecificfluorescenceprobe.Horseradishperoxidasecatalyzesthereactionbetweentheprobeandhydrogenperoxide,whichbindina1:1ratio.Samplesarecomparedtoaknownconcentrationofcholesterolstandardwithinthe96-wellmicrotiterplateformat.Samplesandstandardsareincubatedfor45minutesandthenreadwithastandard96-wellfluorometricplatereader.3.CholesterolAssayPrinciple
SampleTypeSerum,Plasma
DetectionMethodFluorometric
CharacteristicsHDLandLDL/VLDLCholesterolAssayKitisasimplefluorometricassaythatcanmeasuretheamountsofHDLandLDL/VLDLcholesterolpresentinplasmaorserumsampleswithina96-wellmicrotiterplateformat.Theassaywilldetecttotalcholesterol(cholesterylestersplusfreecholesterol)inthepresenceofcholesterolesteraseoronlyfreecholesterolintheabsenceoftheesteraseenzyme.Eachkitprovidessufficientreagentstoperformupto192assays,includingblanks,cholesterolstandardsandunknownsamples.Samplecholesterolconcentrationsaredeterminedbycomparisonwithaknowncholesterolstandard.Cholesterylesterscanbequantifiedbysubtractingthefreecholesterolvaluesfromthetotalcholesterolvalue.
Components
  1. 96-wellMicrotiterPlate:Two96-wellclearbottomblackplates.
  2. CholesterolStandard:One50μLtubeofa10mMcholesterolsolutioninethanol.
  3. AssayDiluent(5X):One100mLbottle.
  4. FluorescenceProbe:One200μLtubeinDMSO.
  5. HRP:Two100μLtubesof100U/mLsolutioneachinglycerol.
  6. LDLPrecipitationSolution(2X):Two20mLbottles.

Box2(shippedonblueicepacks)

Materialnotincluded
  1. Distilledordeionizedwater
  2. 1XPBS
  3. Microcentrifuge
  4. Microcentrifugetubes
  5. 10μLto1000μLadjustablesinglechannelmicropipetteswithdisposabletips
  6. 50μLto300μLadjustablemultichannelmicropipettewithdisposabletips
  7. Multichannelmicropipettereservoir
  8. Fluorescencemicroplatereadercapableofreadingexcitationinthe530-570nmrangeandemissioninthe590-600nmrange.
  9. Superoxidedismutase(optional)5
BackgroundCholesterolisalipidsterolthatisproducedinandtransportedthroughoutthebloodstreamineukaryotes.Cholesterolisacriticalcompoundusedinthestructureofcellmembranes,hormones,andcellsignaling.Itisanessentialcomponentofanimalcellstructureinordertomaintainpermeabilityandfluidity.Cholesterolisaprecursorforsteroidhormones,includingtheadrenalglandhormonescortisolandaldosterone,sexhormonesprogesterone,estrogens,andtestosterone,aswellasbileacids,andvitaminD.Cholesterolistransportedaroundthebodywithinlipoproteins,whicharesubmicroscopicparticlescomposedoflipidandproteinheldtogetherbynoncovalentforces.Theirgeneralstructureisthatofaputativespheroidalmicroemulsionformedfromanouterlayerofphospholipids,unesterifiedcholesterol,andproteins,withacoreofneutrallipids,predominatelycholesterylestersandtriacylglycerols(TAG).Lipoproteinsmainfunctionistotransporttheselipidsaroundthebodyintheblood.Lipoproteinparticleshavehydrophilicgroupsofphospholipids,cholesterol,andapoproteinsdirectedoutward.Suchcharacteristicsmakethemsolubleinthesaltwater-basedbloodpool.Triglyceride-fatsandcholesterolestersarecarriedinternally,shieldedfromthewaterbythephospholipidmonolayerandtheapoproteins.Theinteractionoftheproteinsformingthesurfaceoftheparticleswithenzymesintheblood,witheachother,andwithspecificproteinsonthesurfacesofcellsdeterminewhethertriglyceridesandcholesterolwillbeaddedtoorremovedfromthelipoproteintransportparticles.Lipoproteinshavecell-specificsignalsthatdirectthelipidstheytransporttocertaintissues.Forthisreason,lipoproteinsexistindifferentformswithinthebloodbasedontheirdensity.Theseincludechylomicrons,very-lowdensitylipoproteins(VLDLs),intermediate-densitylipoproteins(IDLs),low-densitylipoproteins(LDLs),andhigh-densitylipoproteins(HDLs).Thehigherthelipidcontentinalipoprotein,thelessdenseitis.Cholesterolexistswithinalipoproteinasafreealcoholandasafattycholesterylester,whichisthepredominantformofcholesteroltransportandstorage.HighbloodlevelsofLDLsareassociatedwithhealthproblemsandcardiovasculardisease.Forthisreason,LDLisoftenreferredtoasthe"badcholesterol."LDLparticlesthataccumulatewithinarteriescanformplaquesovertime,whichcanincreasechancesofastroke,heartattack,orvasculardisease.HDLparticlesareabletoremovecholesterolfromwithinarteriesandtransportitbacktotheliverforre-utilizationorexcretion,whichisthemainreasonwhythecholesterolcarriedwithinHDLparticlesissometimescalled"goodcholesterol."Monitoringcirculatorylevelsofdifferentlipoproteinsiscriticaltothediagnosisoflipidtransportdisorderssuchasatherosclerosis.
ApplicationNotesOptimalworkingdilutionshouldbedeterminedbytheinvestigator.
Comment

  • AllowsyoutoseparateandindependentlytestHDLandLDL/VLDLfractions
  • Suitableforusewithserumorplasma
  • Cholesterolstandardincluded

ReagentPreparation
  • 1XAssayDiluent:WarmtheAssayDiluent(5X)toroomtemperaturepriortousing.DilutetheAssayDiluent(5X)withdeionizedwaterbydilutingthe100 mLDiluentwith400 mLdeionizedwaterfor500 mLtotal.Mixtohomogeneity.Storethe1XAssayDiluentat4 °Cuptosixmonths.
  • CholesterolEsterase:Reconstitutethepowderwith200μLof1XAssayDiluent.Vortexvigorouslyuntildissolved.Preparealiquotsandstoreat-20 °Ctoavoidmultiplefreezethawsofthereconstitutedpowder.
  • CholesterolReactionReagent:PreparethereagentbydilutingtheCholesterolOxidase1:50,HRP1:50,FluorescenceProbe1:50,andCholesterolEsterase1:250in1XAssayDiluent.(eg.For100assays,combine100μLofCholesterolOxidase,100μLofHRP,100μLFluorescenceProbe,and20μLCholesterolEsterasewith1XAssayDiluentto5 mLtotalsolution).Mixthoroughlyandprotectthesolutionfromlight.Forbestresults,placetheCholesterolReactionReagentoniceandusewithin30 minutesofpreparation.DonotstoretheCholesterolReactionReagentsolution.Notes:1.Iftestingfortheconcentrationoffreecholesterolonly,omittheadditionofCholesterolEsterasefromtheCholesterolReactionReagentsolution.2.TheFluorescenceProbeislightsensitiveandmustbestoredaccordingly.
SamplePreparation

Samplesshouldbeusedimmediatelyorstoredat-80 °Cpriortoperformingtheassay.Optimalexperimentalconditionsforsamplesmustbedeterminedbytheinvestigator.Thefollowingrecommendationsareonlyguidelinesandmaybealteredtooptimizeorcomplementtheusersexperimentaldesign.Asetofserialdilutionsisrecommendedforsamplestoachieveoptimalassayresultsandminimizepossibleinterferingcompounds.Runpropercontrolsasnecessary.Alwaysrunastandardcurvewithsamples.•Serum:Collectbloodinatubewithnoanticoagulant.Allowthebloodtoclotatroomtemperaturefor30 minutes.Centrifugeat2500xgfor20 minutes.Removetheserumlayerandstoreonice.Avoiddisturbingthewhitebuffylayer.Aliquotsamplesfortestingandstoreat-80 °C.Performdilutionsin1XAssayDiluent.Cholesterollevelsinserumaverageabout3 %higherinvaluethaninthecorrespondingplasmapair(Ref.2).•Plasma:Avoidhemolyzedandlipemicbloodsamples.Collectbloodwithheparinorcitrateandcentrifugeat2000xgand4 °Cfor10 minutes.Removetheplasmalayerandstoreonice.Avoiddisturbingthewhitebuffylayer.Aliquotsamplesfortestingandstoreat-80 °C.Performdilutionsin1XAssayDiluent.Notes:

  1. SampleswithNADHconcentrationsabove10μMandglutathioneconcentrationsabove50μMwilloxidizetheprobeandcouldresultinerroneousreadings.Tominimizethisinterference,it6isrecommendedthatsuperoxidedismutase(SOD)beaddedtothereactionatafinalconcentrationof40U/mL.
  2. AvoidsamplescontainingDTTorβ-mercaptoethanolsincethefluorescenceprobeisnotstableinthepresenceofthiols(above10μM).PreparationofHDLandLDL/VLDLFractions
    1. Add200μLofsample(serumorplasma)toamicrocentrifugetube.Add200μLofthePrecipitationReagentandmixwellbyvortexing.Allowmixturetoincubate5-10 minutesatroomtemperature(precipitationwilloccur).
    2. Centrifugethemixtureat2000xg(~5000rpm)for20 minutes(pelletshouldbevisible).Slowlyandcarefullytransferthesupernatant(HDLfraction)intoanewtube,leavingthepellet(LDL/VLDLfraction).Resuspendanddissolvethepelletin400μLofPBS,vortexingwell.Ensurethatthepellet(LDL/VLDLfraction)iscompletelydissolvedbeforetesting.
    3. FurtherdilutetheHDLorLDL/VLDLfractionsamples1:25(1:50finaldilution)to1:100(1:200finaldilution)in1XAssayDiluentbeforerunningtheassay.Assayimmediatelyanddonotstoresolutions.Notes:
      1. SampleswithNADHconcentrationsabove10μMandglutathioneconcentrationsabove300μMwilloxidizetheprobeandcouldresultinerroneousreadings.Tominimizethisinterference,itisrecommendedthatsuperoxidedismutase(SOD)beaddedtothereactionatafinalconcentrationof40U/mL.
      2. AvoidsamplescontainingDTTorβ-mercaptoethanolsincethefluorescenceprobeisnotstableinthepresenceofthiols(above10μM).

AssayProcedure

Eachcholesterolstandardandsampleshouldbeassayedinduplicateortriplicate.Afreshlypreparedstandardcurveshouldbeusedeachtimetheassayisperformed.

  1. Add50μLofthedilutedcholesterolstandardsorsamplestothe96-wellmicrotiterplate.
  2. Add50μLofthepreparedCholesterolReactionReagenttoeachwellandmixthewellcontentsthoroughly.
  3. Covertheplatewellstoprotectthereactionfromlight.Incubatetheplatefor45 minutesat37 °C.
  4. IMMEDIATELYreadtheplatewithafluorescencemicroplatereaderequippedforexcitationinthe530-570nmrangeandforemissioninthe590-600nmrange.
  5. CalculatetheconcentrationofcholesterolwithinsamplesbycomparingthesampleRFUtothecholesterolstandardcurve.

CalculationofResults
  1. Calculatetheaveragefluorescencevaluesforeverystandard,control,andsample.Subtracttheaveragezerostandardvaluefromitselfandallstandardandsamplevalues.Thisisthecorrectedfluorescence.
  2. PlotthecorrectedfluorescenceforthestandardsagainstthefinalconcentrationofthecholesterolstandardsfromTable1todeterminethebestcurve.SeeFigure3foranexamplestandardcurve.
  3. Determinethecholesterolconcentrationofthesampleswiththeequationobtainedfromthelinearregressionanalysisofthestandardcurve.Substitutethecorrectedfluorescencevaluesforeachsample.Remembertoaccountforalldilutionfactors.SamplecorrectedfluorescenceTotal,HDL,LDL/VLDL(μM)=xSampledilutionCholesterolSlopeCholesterylEster(μM)=TotalCholesterol-FreeCholesterolTotalCholesterol(Unfractionated)(μM)~HDLCholesterol(μM)+LDL/VLDLCholesterol(μM)Note:FortheconversionofresultsfromμMtomg/dl,dividethecholesterolconcentration(μM)by25.9.
RestrictionsForResearchUseonly
HandlingAdviceAvoidmultiplefreeze/thawcycles.
Storage4°C/-20°C
StorageCommentUponreceipt,storetheCholesterolStandard,FluorescenceProbe,HRP,CholesterolOxidase,andCholesterolEsteraseat-20°C.TheFluorescenceProbeislightsensitiveandmustbestoredaccordingly.Avoidmultiplefreeze/thawcycles.Storetheremainingkitcomponentsat4°C.
SupplierImages
ELISA image for HDL and LDL/VLDL Cholesterol Assay Kit (ABIN2345055)holesterolValuesofHumanSerumTestedUsingtheHDLandLDL/VLDLCholesterolAssay...
Productcitedin:Jang,Bae,Kim,Cho,Yuk,Han,Youn,Kwon,Hwang,Kimetal.:"TheherbalformulaKH-204isprotectiveagainsterectiledysfunctionbyminimizingoxidativestressandimprovinglipidprofilesinaratmodeloferectiledysfunctioninducedby..."in:BMCcomplementaryandalternativemedicine,Vol.17,Issue1,pp.129,2017(PubMed).

Sessions-Bresnahan,Schauer,Heuberger,Carnevale:"EffectofObesityonthePreovulatoryFollicleandLipidFingerprintofEquineOocytes."in:Biologyofreproduction,Vol.94,Issue1,pp.15,2016(PubMed).

Angelovich,Shi,Zhou,Maisa,Hearps,Jaworowski:"Exvivofoamcellformationisenhancedinmonocytesfromolderindividualsbybothextrinsicandintrinsicmechanisms."in:Experimentalgerontology,Vol.80,pp.17-26,2016(PubMed).

Gamal,Sadek,Rashed,Shawky,GamalEl-Din:"Effectofgamma-carboxylaseinhibitiononserumosteocalcinmaybepartiallyprotectiveagainstdevelopingdiabeticcardiomyopathyintype2diabeticrats."in:Diabetes&vasculardiseaseresearch,Vol.13,Issue6,pp.405-417,2016(PubMed).

Maisa,Hearps,Angelovich,Pereira,Zhou,Shi,Palmer,Muller,Crowe,Jaworowski:"MonocytesfromHIV-infectedindividualsshowimpairedcholesteroleffluxandincreasedfoamcellformationaftertransendothelialmigration."in:AIDS,Vol.29,Issue12,pp.1445-57,2015(PubMed).

OHare,Wang,Montasser,Chang,Mitchell,Zaghloul:"DisruptionofldlrcausesincreasedLDL-candvascularlipidaccumulationinazebrafishmodelofhypercholesterolemia."in:Journaloflipidresearch,Vol.55,Issue11,pp.2242-53,2014(PubMed).


新闻动态
行业前沿
技术文章
最新产品

188进口试剂采购网 www.188bio.cn -中国试剂网,试剂网,化学试剂网,国药试剂,抗体公司,试剂公司,试剂盒公司,苏州试剂公司,北京化学试剂公司,天津化学试剂,试剂商城,试剂代理,流式抗体 细胞库查询 sitemap