Description:
REAADSProteinCAntigenisanenzyme-linkedimmunosorbentassay(ELISA)forthequantitativedeterminationofProteinCAntigenincitratedhumanplasma.
KitComposition:
Reagents
- 12x8anti-humanProteinCantibodycoatedmicrowells.
- 60mlSampleDiluent(blue-greensolution);containssodiumazide.
- 3vialsx0.5mllyophilizedReferencePlasma,withassaysheet
- 12mlanti-humanProteinCHRPConjugate(bluesolution).
- 13mlSubstrate(TMBandH2O2).
- 15mlStoppingSolution(0.36Nsulfuricacid).
- 30mlWashConcentrate(33Xphosphatebufferedsalinewith0.01%Tween20).Note:turbiditymayappearinwashconcentratewhichwillnotaffectcomponentperformanceandshoulddisappearwhenworkingdilutionisprepared.
Storeat2–8°C.DoNotFreeze.
Materialsrequiredbutnotsupplied
- ProteinCControlPlasma.ReconstituteControlPlasmaselectedforusefollowingmanufacturer’sinstructions,andstoreasrecommended.
- Reagentgradewater(1L)topreparePBS/Tween20washsolution,toreconstituteReferencePlasma,andtozeroorblanktheplatereaderduringthefinalassaystep.
- Graduatedcylinders
- Precisionpipettorscapableofdeliveringbetween5and1000microliters,withappropriatetips
- Miscellaneousglasswareappropriateforsmallvolumehandling
- Flaskorbottle,1liter
- Washbottles,preferablywiththetippartiallycutbacktoprovideawidestream,oranautomatedorsemi-automatedwashingsystem
- Disposablegloves,powder-freerecommended
- PlatereADIngspectrophotometercapableofreadingabsorbanceat450nm(witha650nmreferenceifavailable)
- Multichannelpipettorscapableofdeliveringto8wellssimultaneously
- Microdilutiontubesforpatientsamplepreparation
MeasurementPrinciple:
Principle
TheProteinCAntigenassayisasandwichELISA.AcaptureantibodyspecificforhumanProteinCiscoatedto96-microwellpolystyreneplates.Dilutedpatientplasmaisincubatedinthewells,allowinganyavailableProteinCtobindtotheanti-humanProteinCantibodyonthemicrowellsurface.Theplatesarewashedtoremoveunboundproteinsandotherplasmamolecules.BoundProteinCisquantitatedusinghorseradishperoxidase(HRP)conjugatedanti-humanProteinCdetectionantibody.Followingincubation,unboundconjugateisremovedbywashing.Achromogenicsubstrateoftetramethylbenzidine(TMB)andhydrogenperoxide(H2O2)isaddedtodevelopacoloredreaction.Theintensityofthecolorismeasuredinopticaldensity(O.D.)unitswithaspectrophotometerat450nm.ProteinCAntigenrelativepercentconcentrationsinpatientplasmaaredeterminedagainstacurvepreparedfromthereferenceplasmaprovidedwiththekit.
Procedure
DilutedcitratedpatientplasmaandcontrolsareincubatedinmicrowellscoatedwithcaptureantibodyspecificforhumanProteinC.Duringanincubationperiod,patientProteinCisallowedtobindtothesurface.
Followingawashtoremoveanyunboundplasma,horseradishperoxidase(HRP)conjugatedanti-humanProteinCdetectionantibodyisaddedtothewells.Afterwashingtoremoveunboundconjugate,achromogenicsubstrateisadded,resultinginasolublecoloredproductthatismeasuredinaspectrophotometerat450nmfollowingtheadditionofastopsolution.
PatientProteinClevelsaredeterminedfromasixpointcurvepreparedfromthereferenceplasmaprovidedinthekit.Totalincubationtimeis60minutes.
AssayProcedure:
1.Removeanymicrowellstripsthatwillnotbeusedfromtheframeandstoretheminthebag provided.
2.Assayeachreferenceplasmadilutioninduplicate.Duplicatedeterminationsarealsorecommended forpatientandcontrolsamples.Onewellshouldberunasareagentblank;samplediluentwithout serumisaddedtothewellasexplainedinstep7ofthissection.Thiswellistreatedthesameasa controlorpatientsampleinsubsequentassaysteps.Awaterblankwellshouldbeincludedwith eachplate;itistoremainemptyuntil200µlofreagentgradewaterisaddedatthecompletionof theassay,immediatelypriortoreadingtheplate.Thewaterblankwellistobeusedtozerotheplate reader.
3.Pre-diluteallplasmas(1:2dilutioninSampleDiluent)asfollows: Referenceplasma:add100µlreferenceplasmato100µlSampleDiluent Controlandpatientsamples:add20µlplasmato20µlSampleDiluent Mixwell.Thesepre-dilutionsareutilizedinpreparingtheworkingdilutionsinsteps4and5.
4.Usingthe1:2referenceplasmadilutionfromstep3,preparesixworkingreferencedilutionsas describedbelow.
VolumeReference Plasma(1:2) | VolumeSample Diluent | *ReferenceLevel | ||
30μl | + | 500μl | = | 150 |
20μl | + | 500μl | = | 100 |
15μl | + | 500μl | = | 75 |
10μl | + | 500μl | = | 50 |
10μl | + | 1000μl | = | 25 |
10μl | + | 2000μl | = | 12.5 |
*Referencelevelvaluetobeusedforconstructingreferencecurveonly |
5.Prepareworkingdilutionsofcontrolandpatientsamplesbyadding20µlofpredilutedplasma(1:2 dilutionfromstep3)to500µlSampleDiluent.(Note:thesedilutionscorrespondtothe100% referenceplasmadilution.)
6.Mixthoroughly,andadd100µloftheworkingdilutions(referenceplasmas,controlsandpatient samples)totheappropriatemicrowells.
7.Add100µlofSampleDiluenttothereagentblankwell.Leavethewaterblankwellempty.
8.Incubate40minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthe microwellsanddumpthesamplefluid.Donotallowsamplestocontaminateothermicrowells.
9.Wash4timeswithworkingwashsolution(PBS/Tween20).Eachwellshouldbefilledwithwash solutionperwash.Washsolutionintheemptywellintendedtoserveasawaterblankwillnot interferewiththeprocedure.Invertmicrowellsbetweeneachwashtoemptyfluid.Useasnapping motionofthewristtoshaketheliquidfromthewells.Theframemustbesqueezedatthecenteron thetopandbottomtoretainmicrowellmodulesduringwashing.Blotonabsorbentpapertoremove residualwashfluid.Donotallowwellstodryoutbetweensteps.
10.Add100µlConjugate(blue)toeachwell(exceptthewaterblankwell).
11.Incubatefor10minutesatroomtemperature.Aftertheincubationiscomplete,carefullyinvertthe microwellsanddumptheconjugatesolution.
12.Wash4timeswithworkingwashsolution(PBS/Tween20)asinstep9.Washsolutioninthewater blankwelldoesnotinterferewiththeprocedure.Useasnappingmotiontodraintheliquid,andblot onabsorbentpaperafterthefinalwash.Donotallowthewellstodryout.
13.Add100µlSubstratetoeachwell(exceptforthewaterblankwell)andincubatefor10minutesat roomtemperature.Addthesubstratetothewellsatasteadyrate.Bluecolorwilldevelopinwells withpositivesamples.
14.Add100µlStoppingSolution(0.36Nsulfuricacid)toeachwell(exceptforthewaterblankwell)to stoptheenzymereaction.BesuretoaddStoppingSolutiontothewellsinthesameorderandatthe samerateastheSubstrateSolutionwasadded.BlueSubstratewillturnyellowandcolorlesssubstrate willremaincolorless.DonotaddStoppingSolutiontothewaterblankwell.Instead,add200µlof reagentgradewatertothewaterblankwell.Blankorzerotheplatereaderagainstthewaterblank well.ReadtheO.D.ofeachwellat 450nm,againsta650nmreferencefilter(ifavailable).Forbestresults,theO.D.valuesshouldbemeasuredwithin30minutesaftertheadditionofStoppingSolution.
Performance:
ClinicalPerformance
PlasmasamplesfromhealthyblooddonorsandfrompatientswithahistoryofthrombosisweretestedtodefineandcomparetheclinicalperformanceofREAADSProteinCELISAwithawellestablished,commerciallyavailableProteinCAntigenRocketEIDmethod.Asshowninthetable,theresultscorrelatedwell,andwereshowntobestatisticallysimilarbysinglefactorAnova.
TechnicalPerformance
Intra-assayprecisionofREAADSProteinCELISAis7.0%whileintra-assayprecisionis7.5%Linearity,expressedasthecoefficientofdetermination(r2)is0.992withameanaccuracyof99.4%REAADSProteinCELISAisarapid,convenient,highlyaccurateandprecisemethodforthequantitativedeterminationofProteinClevelsinhumanplasma.
Background:
ProteinCisavitaminK-dependentproteinsynthesizedprimarilybyhepatocytesintheliverandplaysanimportantphysiologicroleintheProteinCAnticoagulantSystem.ProteinC,thrombinfrombloodclots,andendothelialcells,throughcomplexinteractionswithotherfactorsofthecoagulationcascade,contributetothemaintenanceofnormalhemostaticmechanismsbydown-regulatingclotformationandbypromotingfibrinolysis.TheProteinCAnticoagulantSystemisactivatedbythebindingofthrombintothrombomodulin,atransmembraneproteinreceptoronendothelialcells.Thethrombin-thrombomodulinbindingonendothelialcellmembranesactivatescirculatingProteinC.ActivatedProteinCbindstoProteinSonthemembraneofendothelialcellsorplatelets.InthisProteinC-ProteinScomplex,activatedProteinCisnowcapableofinactivatingcoagulationfactorsVaandVIIIa,down-regulatingclotformation.ActivatedProteinCalsoenhancesthefunctionoftissueplasminogenactivator(TPA)bydissociatingthismoleculefromitsinhibitor,plasminogenactivatorinhibitor-1(PAI-1),therebyfacilitatingclotdissolutionorfibrinolysis.
ProteinCdeficiency,eithercongenitaloracquired,mayleadtoseriousthromboticeventssuchasthrombophlebitis,deepveinthrombosis,orpulmonaryembolism.Patientswithacongenitalheterozygousdeficiencymaypresentwithvenousthrombosisinyoungadulthood,whilepatientswiththerarehomozygousdeficiencypresentwithmassivethrombosis(purpurafulminans)duringtheneonatalperiod.TheprevalenceofProteinCdeficiencyinthegeneralpopulationhasbeenestimatedat1in300.Inyoungerpatients(<40-45years)withrecurrentvenousthrombosis,thefrequencyofProteinCdeficienciesmaybeashighas10to15%.AcquiredProteinCdeficiencymaybeseeninliverdisease,extensivethromboticepisodes,surgery,oralanticoagulanttherapy,antiphospholipidsyndrome,etc.AdecreasedProteinCactivityinplasmamaybetheresultoflowconcentrationsandfunction(typeI)oronlylowfunction(typeII).
ThelaboratorydiagnosisofProteinCdeficiencymayrequirebothquantitativeandqualitative(functional)determinations.QuantitativedeterminationsofProteinCAntigenarebasedonimmunologicproceduressuchasradialimmunodiffusioningel,Laurellrocketimmunoelectrophoresisandenzyme-linkedimmunosorbentassay(ELISA).ELISAproceduresarelesslaborintensiveandofferseveraladvantagesincludingmoreobjective,accurateandreproducIBLeresults.Inaddition,ELISAallowsautomationwithcommonlyavailablelaboratoryinstruments.
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ldh试剂盒指乳酸脱氢酶检测试剂盒,是一种基于diaphorase催化的INT显色反应,通过比色法检测细胞毒性时释放的乳酸脱氢酶活性或检测其它样品中的乳酸脱氢酶活性的试剂盒。可以用于常规的乳酸脱氢酶活性的检测,更常用于以LDH释放为指标的细胞毒性检测。
ctl毒性杀伤检测试剂盒是基于LDH在胞浆内含量丰富,正常时不能通过细胞膜,当细胞受损伤或死亡时可释放到细胞外,此时细胞培养液中LDH活性与细胞死亡数目成正比,用比色法测定并与靶细胞对照孔LDH活性比较,可计算效应细胞对靶细胞的杀伤。
百奥森18分钟霉菌毒素检测试剂盒,有需要的可以了解下!多种规格型号可供选择
产品名称
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1、 配制JC-1染色工作液:
取适量JC-1 Stain (200×),按照每50μl JC-1 Stain (200×)加入8ml ddH2O的比例稀释JC-1,剧烈Vortex充分溶解并混匀JC-1。然后再加入2ml JC-1 Buffer(5×),混匀后即为JC-1染色工作液。6孔板每孔所需JC-1染色工作液的量为1ml,其它培养器皿的JC-1染色工作液的用量以此类推。
2、 设置阳性对照:
推荐CCCP(10mM)加入到细胞培养液中处理细胞。随后按照下述方法装载JC-1,进行线粒体膜电位的检测。对于特定的细胞,CCCP的作用浓度和作用时间可能有所不同,需自行参考相关文献资料确定。
3、对于悬浮细胞:
a. 取1~6×105细胞,重悬于0.5ml细胞培养液中,细胞培养液中可以含血清和酚红。
b. 加入0.5ml JC-1染色工作液,颠倒数次混匀。细胞培养箱中37℃孵育。
c. 在孵育期间,按照每1ml JC-1 Buffer(5×)加入4ml蒸馏水的比例,配制适量的JC-1 Buffer(1×),并放置于冰浴。
d. 37℃孵育结束后, 4℃ 600g离心3~4min,沉淀细胞。弃上清,注意尽量不要吸除细胞。
f. 再用JC-1 Buffer(1×)重悬后,用荧光显微镜或激光共聚焦显微镜观察,也可以用荧光分光光度计检测或流式细胞仪分析。
4、对于贴壁细胞:
注意:对于贴壁细胞,如果希望采用荧光分光光度计或流式细胞仪检测,应先收集细胞,重悬后参考悬浮细胞的检测方法。
a.吸除6孔板培养液,根据具体实验如有必要可以用PBS或其它适当溶液洗涤细胞一次,加入1ml细胞培养液。细胞培养液中可以含有血清和酚红。
b. 加入1ml JC-1染色工作液,充分混匀。细胞培养箱中37℃孵育。
c. 在孵育期间,按照每1ml JC-1 Buffer(5×)加入蒸馏水的比例,配制适量的JC-1 Buffer(1×),并放置于冰浴。
d. 37℃孵育结束后, 吸除上清,用JC-1 Buffer(1×)洗涤2次。
e. 加入2ml细胞培养液,培养液中可以含有血清和酚红。
f. 荧光显微镜或激光共聚焦显微镜下观察。
5、对于纯化的线粒体:
a. 把配制好的JC-1染色工作液再用JC-1 Buffer(1×)稀释5倍。
b. 0.9ml 5倍稀释的JC-1染色工作液中加入0.1ml总蛋白量为10~100μg纯化的线粒体。
c. 用荧光分光光度计或荧光酶标仪检测:混匀后直接用荧光分光光度计进行时间扫描,激发波长为485nm,发射波长为590nm。如果使用荧光酶标仪,激发波长不能设置为485nm时,可以在475~520nm范围内设置激发波长。另外,也可以参考下面步骤6中的波长设置进行荧光检测。
d. 用荧光显微镜或激光共聚焦显微镜观察:方法同下面的步骤6。
6、荧光观测和结果分析:
检测JC-1单体时可以把激发光设置为490nm,发射光设置为530nm;检测JC-1聚合物时,可以把激发光设置为525nm,发射光设置为590nm。出现红色荧光说明线粒体膜电位比较正常,细胞的状态也比较正常。
注意事项:
1、 JC-1 Stain(200×)应完全溶解混匀后使用,但应避免反复冻融。必须先把JC-1 Stain(200×)用ddH2O充分溶解混匀后,才可加入JC-1 Buffer(1×)。不可先配制JC-1 Buffer(1×)再加入JC-1 Stain(200×),否则导致JC-1很难充分溶解,严重影响后续的检测。
2、 对于6孔板中的样品,本试剂盒共可以检测100个样品;对于12孔中的样品,本试剂盒共可以检测200个样品。
3、 装载完JC-1后用JC-1 Buffer(1×)洗涤时,尽量使JC-1 Buffer(1×)保持4℃左右,此时的洗涤效果较好。
4、 勿把JC-1 Buffer(5×)全部配制成1×,因为操作过程中需直接使用JC-1 Buffer(5×)。
5、 如JC-1 Buffer(5×)中有沉淀,必须全部溶解后才能使用,为促进溶解可以在37℃加热。
6、 CCCP为线粒体电子传递链抑制剂,有一定毒性,请注意小心防护。
实验室要开展支原体检测,方法是PCR法,先要采购试剂盒,用过的同学给推荐一下好用的品牌呗
牛胰岛素,是一种多肽
在1965年9月17日我国完成了结晶牛胰岛素的全合成。经过严格鉴定,它的结构、生物活力、物理化学性质、结晶形状都和天然的牛胰岛素完全一样。这是世界上第一个人工合成的蛋白质,为人类认识生命、揭开生命奥秘迈出了可喜的一大步。这项成果获1982年中国自然科学一等奖。
1953年,英国人F. SangerSanger由于测定了牛胰岛素的一级结构而获得1958年诺贝尔化学奖。
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