Description:
ChromogenixS-2765™isachromogenicsubstratefordeterminationofFactorXaactivity.Itisalsoverysensitivetotrypsin.
S-2765™ issuitable formeasuringFXainhibition inheparinanti-Xaassaysandantithrombinanti-Xaassays.
Composition:
EachvialcontainsthechromogenicsubstrateS-2765™,25mgandmannitol60mgasabulkingagent.
StABIlity:Lyophilizedsubstance:stableat25°Cuntilexpirydateprintedonthelabel.Thesubstanceishygroscopicandshouldbestoredinadryplace.
Solution:2mmol/LinH2Oisstableforsixmonthsat2to8°C.Suitablestocksolution:1-2mmol/LinH2O.
Chemicalname:N-a-Benzyloxycarbonyl-Darginyl-L-glycyl-L-arginine-pnitroaniline-dihydrochloride
Formula:N-a-Z-D-Arg-Gly-Arg-pNA·2HCl
Mol.wt.:714.6
FactorXaIUandEnzymeActivity:
FactorXa,whichhasamolecularweightof44KDa,istheactivatedformofFactorX(MW:59KDa).
TheInternationalUnitsofFactorXcorrespondtotheamountofFactorXcontainedin1mlofnormalplasma.Thisisabout8mg/lor0.13µmol/l.
SincethereisnoWHOstandardforFXa,onewouldassumethatifalltheFactorXinnormalplasmawasconvertedtotheactivatedform,theFactorXaconcentrationwouldbeapproximately5.7mg/l.
TheactivityofhumanFactorXaascalculatedfromthekinetictablesis1.5nkat/µgwiththechromogenicsubstrateS-2222™,and4.4µkat/µgwiththechromogenicsubstrateS-2765™.
Theactivityof1µgofFactorXaasdeterminedbyFrieberger(1)is1.9nkatchromogenicsubstrateS-2222™.
Thus,1plasmaequivalentunitofFactorXwouldcorrespondto15.2nkatchromogenicsubstrateS-2222™.
FribergerPetal.
Syntheticpeptidesubstrateassaysandfibrinolysisandtheirapplicationonautomates.In:SeminarsinThrombosisandHaemostasis,Vol.9,281-300(1983).
FactorXMethod:
DeterminationoffactorXinplasmawithChromogenicSubstrateS-2765™
MeasurementPrinciple
Themethodisbasedonatwo-stageprinciple.Instageone,FactorXisactivatedinthepresenceofcalciumtoFactorXa(FXa)usingtheactivatorRussell’sVipervenom(RVV).Instagetwo,thegeneratedFXahydrolysesthechromogenicsubstrateZ-D-Arg-Gly-Arg-pNA(S-2765™),thusliberatingthechromophoricgrouppNA(p-nitroaniline).Thecoloristhenreadphotometricallyat405nm.ThegeneratedFXa(andthustheintensityofcolor)isproportionaltotheFXactivityofthesample.
FactorX | RVV | FactorXa |
Z-D-Arg-Gly-Arg-pNA+H2O | FXa | Z-D-Arg-Gly-Arg-OH+pNA |
Reagents
- S-2765™,25mgArt.No.S821413
ReconstitutethesubstrateS-2765™(MW:714.6)with20mlsterilewater. - Russell’sViperVenom(RVV)
PrepareasolutionofRussell’sViperVenomataconcentrationof0.087mg/ml. - CaCl2
0.1mol/lcalciumchloridesolution. - TrisEDTABuffer
Dilutethebuffer1:10withdistilledwateraccordingtotheinsertsheetinstructions. - NormalPlasma
Calibrated,lyophilizedorfreshfrozenhumanplasmaisusedforthestandardizationoftheassay.Apoolednormalplasmacanbepreparedbytakingsamplesfrom20healthydonors.10-30mlcitrateblood(9volbloodand1vol0.1mol/lsodiumcitrate)fromeachdonoriscentrifugedat2000xgfor20minutesat15-25°C.Theplasmaispooledandsubsequentlydispensedinsmallvolumes,whicharefrozenrapidlyat-20°Corbelow.Toavoidlowtemperatureactivationofprekallikreintheplasmaiskeptat15-25°Cbeforeuseorfreezing.Thawingofplasmashouldbeperformedat37°Candthenkeptat15-25°Cuntilused. - RVV+CaCl2
Beforeuse,mix1volumeofRVVwith1volumeofCaCl2.Themixtureisstablefor48hoursat2-8°C. - Aceticacid20%
Aceticacidisusedasastoppersolutionintheend-pointmethod.
Specimencollection
Blood(9vol)ismixedwith0.1mol/lsodiumcitrate(1vol)andcentrifugedat2000xgfor20minutesat20-25°C.Storage:1weekat2-8°Cor3-4monthsat-20°C.
Standardcurve
Predilution | FinalDilution | |||
FX% | NormalPlasma ml | Buffer ml | Predilplasma ml | Buffer ml |
0 | – | – | – | 1000 |
25 | 25 | 75 | 50 | 1000 |
50 | 50 | 50 | 50 | 1000 |
75 | 75 | 25 | 50 | 1000 |
100 | – | – | 50 | 1000 |
124 | – | – | 50 | 800 |
Method
SampleDilution | |
Buffer | 1000 |
Testplasmaorstandard | 50 |
Mix |
AcidStoppedMethod | A | B |
DilutedSample | 200 ml | 50 ml |
Incubateat37°C | 3-4min | 3-4min |
Substrate(37°C) | 200 ml | 50 ml |
Mixandaddwithin30sec | ||
RVV+ CaL2 | 200 ml | 50 ml |
Mixandincubateat37°C | 3min | 3min |
Aceticacid20% | 200 ml | 50 ml |
A=testtubemethod
B=microplatemethod
Sampleblankactivitiesshouldbedeterminedandsubtractedwhenanalyzingstronglycoloredplasma,e.g.lipemicandhemolytic.Thesampleblanksarepreparedbymixingthedilutedsample,aceticacid20%andwaterinsteadofthereagents(400µlfortesttubesand100µlformicroplates).Readtheabsorbanceofthesamplesandblanksat405nm.Thecolorisstableforatleastfourhours.WhenpossIBLe,useadualwavelengthmodewith490nmasthereferencewavelength.
Initialratemethod
Whenperformingtheinitialratemethod,transferthemicroplatetoamicroplatereaderimmediatelyaftertheadditionofRVV+CaCl2andreadthechangeinA/min.Themicroplatereadermustbepre-incubatedat37°C.
Calculation
PlotAorΔA/minforthestandardsagainsttheirconcentrationofFactorX.ReadtheFactorXvalueforthecorrespondingAorΔA/minoftheunknowntestsamplefromthestandardcurve.
Bibliography
- KieselWetal.FactorXactivatingenzymefromRussell’sViperVenom;isolationandcharacterisation.Biochemistry15,4901-4905(1976).
- LindhoutMJetal.ActivationofdecarboxyfactorXbyaproteinfromRussell’sViperVenom.PurificationandpartialcharacterisationofactivateddecarboxyfactorX.BiochemBiophysActa533,327-341,(1978).
- BergströmKandEgbergN.DeterminationofvitaminKsensitivecoagulationfactorsinplasma.Studiesonthreemethodsusingsyntheticchromogenicsubstrates.ThrombRes12,531-547(1978).
- VanWijkEMetal.ArapidmanualchromogenicfactorXassay.ThrombRes22,681-686(1981).EgbergNandHeedmanPA.SimplifiedperformanceofamidoliticfactorXassay.ThrombRes25,437-440(1982).
- ChabbatJetal.AprotininisacompetitiveinhibitorofthefactorVIIa-tissuefactorcomplex.ThrombRes71,205-215(1993).
- MielickiWPandGordonSG.ThreestagechromogenicassayfortheanalysisofactivationpropertiesoffactorXbycancerprocoagulant.BloodCoagulFibrinol4,441-446(1993).
- KoppakaVetal.SolublephospholipidsenhancefactorXa-catalyzedprothrombinactivationinsolution.Biochemistry35,7482-7491(1996).
- RiesbeckKetal.HumantissuefactorpathwayinhibitorfusedtoCD4bindsbothFXaandTF/FVIIaatthecellsurface.ThrombHaemost78,1488-1494(1997).
- RomischJetal.Comparativeinvitroinvestigationofprothrombincomplexconcentrates.SeminThrombHemost24,175-181(1998)
- FariaF,etal.AnewfactorXainhibitor(lefaxin)fromtheHaementeriadepressaleec.ThrombHaemost82,1469-73(1999).
ebiomall.com
>
>
>
>
>
>
>
>
>
>
>
>
2.NaHCO3,Ba(OH)3,H2SO4
3.HCL,NaAlO2,NaHSO4
4.Ca(OH)2,Na2CO3,BaCO3
谢谢了
要原因
请问有什么方法检测细胞内活性氧更好,各位有什么经验,及相关试剂盒推荐一下。
A.H2SO4Na2SO4AgNO3BaCl2.
B.NaOHNa2CO3NaHSO4MgCl2
C.CaCl2NaNO3MgSO4BaCl2
D.HNO3KOHKClK2CO3
请问下有无同学需要H37RA的?我是做EAE模型的,上个月购买了BDDifco公司的H37RA(货号),因为购买的时候只能整盒6支购买,但我们用不了那么多,所以想问问有无同学需要的,100mg/支,800元/支或用等价试剂交换。地址广州。有需要的请私信,谢谢!
(1)优级纯试剂 亦称保证试剂,为一级品,纯度高,杂质极少,主要用于精密分析和科学研究,常以GR表示。
(2)分析纯试剂 亦称分析试剂,为二级品,纯度略低于优级纯,杂质含量略高于优级纯,适用于重要分析和一般性研究工作,常以AR表示。
(3)化学纯试剂 为三级品,纯度较分析纯差,但高于实验试剂,适用于工厂、学校一般性的分析工作,常以CP表示。
(4)实验试剂 为四级品,纯度比化学纯差,但比工业品纯度高,主要用于一般化学实验,不能用于分析工作,常以 LR表示。
以上按试剂纯度的分类法已在我国通用。根据化学工业部颁布的“化学试剂包装及标志”的规定,化学试剂的不同等级分别用各种不同的颜色来标志,见表1。
表1 我国化学试剂的等级及标志
bhclna2so4nano3na2co3
chclnaohna2co3nacl
dba(oh)2nahco3alcl3nahso4
暂无品牌问答