Overview | PrinterFriendlyVersion |
Ex/Em(nm) | 490/515 |
MW | N/A |
CAS# | N/A |
Solvent | N/A |
Storage | F/D/L |
Category | ProteinBiochemistry Generalproteins |
Related | BiochemicalAssays |
1.Prepare500XMaleimideGreen™stocksolution:
Add20µLofDMSO(ComponentE)intotheMaleimideGreenvial(ComponentA)tomake500Xstocksolution.
Note:10µLofthestocksolutionisenoughforone96-wellplate.TheunusedMaleimideGreen™stocksolutionshouldbedividedintosingleusealiquots,storedat-20oCandkeptfromlight.
2.Prepare20Xmaleimidereactionmixture:
Add10µLof500XMaleimideGreen™stocksolution(fromStep1)into250µLReactionBuffer(fromComponentB),andmixthemwell.Incubatethe20Xmaleimidereactionmixtureatroomtemperaturefor30min,protectedfromlight.
Note1:Itisveryimportanttoincubatethe20Xmaleimidereactionmixtureatroomtemperatureforatleast30mintomaximizethesignaltobackgroundratio.
Note2:YoushouldseetheyellowcolorafteraddingtheMaleimideGreen™stocksolutionintoreactionbuffer.
3.Preparemaleimideassaymixture:
Addthewholecontentsof20Xmaleimidereactionmixture(260µLfromStep2)into5mLofassaybuffer(ComponentC),andmixwell.
Note:Thismaleimideassaymixtureisnotstable.Usewithin1hour.
4.PrepareserialdilutionsofN-ethylaleimidestandard(0to10μM):
4.1 Add10μLof10mM(10nmol/µL)N-ethylmaleimidestandardstocksolution(ComponentD)to990µLofassaybuffer(ComponentC)togenerate100µM(100pmol/µL)N-ethylmaleimidestandardsolution.
Note:Theunused10mMN-ethylmaleimidestandardsolutionshouldbedividedintosingleusealiquotsandstoredat-20oC.
4.2 Take200μLof100μMN-ethylmaleimidestandardsolution(fromStep4.1)toperform1:3serialdilutionstoget30,10,3,1,0.3,0.1and0μMserialdilutionsofN-ethylmaleimidestandard.
4.3 AddN-ethylmaleimidestandardsandmaleimide-containingtestsamplesintoasolidblack96-wellmicroplateasdescribedinTables1and2
Table1.LayoutofN-ethylmaleimidestandardsandtestsamplesinasolidblack96-wellmicroplate
BL | BL | TS | TS | …. | …. |
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MS1 | MS1 | …. | …. | …. | …. |
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MS2 | MS2 |
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MS3 | MS3 |
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MS4 | MS4 |
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MS5 | MS5 |
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MS6 | MS6 |
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MS7 | MS7 |
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Note:MS=N-ethylmaleimideStandards,BL=BlankControl,TS=TestSamples.
Table2.Reagentcompositionforeachwell
N-ethylmaleimideStandard | BlankControl | TestSample |
SerialDilutions*(50μL) | AssayBuffer:50μL | 50μL |
*Note:AddtheserialdilutionsofN-ethylmaleimidestandardfrom0.1μMto100μMintowellsfromMS1toMS7induplicate.
5.Runmaleimideassay:
5.1 Add50μLofmaleimideassaymixture(fromStep3)toeachwelloftheN-ethylmaleimidestandard,blankcontrol,andtestsamples(seeStep4.3)tohavethetotalmaleimideassayvolumeof100µL/well.
Note:Fora384-wellplate,add25μLofsampleand25μLofmaleimidereactionmixtureintoeachwell.
5.2 Incubatethereactionmixturefor5to30minutesatroomtemperature,protectedfromlight.
Note:Forbestresults,thefluorescenceintensityshouldbereadwithin30minutesduetothefactthatthefluorescencebackgroundincreaseswithtime.
5.3 MonitorthefluorescenceincreasewithafluorescenceplatereaderatEx/Em=490/520nm.
References&Citations | CitationExplorer |
Conjugationwithaninulin-chitosanadjuvantmarkedlyimprovestheimmunogenicityofMycobacteriumtuberculosisCFP10-TB10.4fusionprotein
Authors:WeiliYu,TaoHu
Journal:MolecularPharmaceutics(2016)
ModeratePEGylationofthecarrierproteinimprovesthepolysaccharide-specificimmunogenicityofmeningococcalgroupApolysaccharideconjugatevaccine
Authors:TingtingZhang,WeiliYu,YanfeiWang,TaoHu
Journal:Vaccine(2015):3208--3214
Rapidquantificationofmaleimideinbioconjuatedsamplesusinganovelcolorimetricmethod(TECH2P.761)
Authors:JinfangLiao,HaitaoGuo,ZhenLuo,QinZhao,JackDiwu
Journal:TheJournalofImmunology(2015):206--7
Successfulacquisitionofaneutralizingmonoclonalantibodyagainstanovelneutrophil-activatingpeptide,mitocryptide-1
Authors:TatsuyaHattori,KentaNakashima,TakayukiMarutani,YoshiakiKiso,YoshisukeNishi,HidehitoMukai
Journal:Biochemicalandbiophysicalresearchcommunications(2015):54--59
CD133-targetedpaclitaxeldeliveryinhibitslocaltumorrecurrenceinamousemodelofbreastcancer
Authors:SureshKumarSwaminathan,EmilieRoger,UdayaToti,LinNiu,JohnROhlfest,JayanthPanyam
Journal:JournalofControlledRelease(2013):280--287
PEGasaspacerarmmarkedlyincreasestheimmunogenicityofmeningococcalgroupYpolysaccharideconjugatevaccine
Authors:QingruiHuang,DongxiaLi,AijunKang,WenqiAn,BeiFan,XiaoweiMa,GuanghuiMa,ZhiguoSu,TaoHu
Journal:JournalofControlledRelease(2013):382--389
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定性ELISA只能粗略表示样本待测物含量在某个特定值以上或以下,定性ELISA通常设置阳性对照(P)、和阴性对照(N),结果分别用“阴性”和“阳性”表示。ELISA定性测定的“阴性”和“阳性”的判断标准是试剂盒所确定的阳性判断值,即cut-off值。
定量ELISA是通过一系列不同已知浓度的标准品所对应的OD值做出标准曲线,然后将样本的OD值代入标准曲线,计算出样本中待测物的含量。百特纯大分子Meretciel的定量ELISA试剂盒还可以。
临床实验室对商品定量试剂盒分析性能的验证.pdf(17431.6k)
谢谢谢
3.逆转录:逆转录试剂盒(或者一步法试剂盒),这一步可以用普通PCR做,也可以用水域做。
4.荧光定量PCR试剂:通常有用SYBR Green Mix做的,但是这里建议你用EvaGreen做,灵敏度和平行性都要好于SYBR Green,并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他:除了以上的那些还需要离心管、PCR管或板(Axygen反应比较好)、移液枪等,暂时就想到这么多。
试剂盒里有详细的说明书,告诉你样品需要多少量,每个试剂需要加入多少量,和详细的实验步骤,一般买来就可以用,不用人教。
所以你问一个样需要多少量是没法回答的,测定过程是要加很多种试剂的。
LOQ-定量限LOD-检测限
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