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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Biotin-X NTA [Biotin-X nitrilotriacetic acid, potassium salt] *CAS 856661-92-2*/3009/1 mg
产品编号:3009
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AAT Bioquest/Biotin-X NTA [Biotin-X nitrilotriacetic acid, potassium salt] *CAS 856661-92-2*/3009/1 mg
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Ex/Em(nm)None/None
MW715.98
CAS#856661-92-2
SolventDMSO
StorageF/D
CategoryBiotinandItsDerivatives
BiotinConjugates
RelatedBiochemicalAssays
Biotin-Xnitrilotriaceticacid(biotin-XNTA)iswidelyusedtodetecthistidine-taggedproteinsimmobilizedonnitrocellulosemembranes.TheNTAmoietyofbiotin-XNTAchelatesNiionthatisalsochelatedwithhistidinetags.TheNTA-polyHis-complexcanbedetectedusingstandardenzyme-linkedstreptavidinmethods.Biotin-XNTAcanbeusedtodetectlessthan0.1pmolofhistidine-taggedproteinusingastreptavidin"horserADIshperoxidaseconjugateandchemiluminescencetechniques.Biotin-XNTAcanberemovedfromthehistidine-taggedproteinatpH4.8,allowingtheblottobereanalyzedwithanotherprobe.Incombinationwithfluorescentavidinconjugates,thisNTAbiotinderivativecanbeusedfordetectingpolyhistidine-containingbiomoleculessuchasfusionproteins.ThisNTABiotinderivativeisabifunctionalreagentthatisusedtodetecthistidine-taggedproteinsimmobilized.ThenitrilotriaceticacidisusedtochelateaNi(II)ionatfourofitssixcoordinationsites.Theremainingtwositesareavailableforbindingtoahistidinetag.Thebiotinfunctionalgroupcanthenbedetectedusingastreptavidin-horseradishperoxidaseconjugateandchemiluminescence.Usingthisbiotinylatednitrilotriaceticacid,itispossIBLetodetectlessthan0.11pmolofhistidine-taggedEscherichiacoliRNApolymerasesigma70subunit.ThisreagentisalsoabletospecificallydetectHis-taggedsigma70fromawholecelllysatefollowingSDS-PAGEandtransfertonitrocellulose.ThereagentcanbedissociatedfromtheHis-taggedproteinatpH4.8andtheblotcanbereprobedwithamonoclonalantibodyfordetectionofdifferentproteinsonthesameblot.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

SampleProtocolfordetectionofHis-tagged ProteininPVDF:

1)                  PrepareyourPVDFblotsasneeded(blocknonspecificbindingsitesandwashtheblot).

2)                  DissolvetheBiotin-XNTAinDMSOorH2Oatconcentrationof1mg/mL

3)                  PreparefreshStainingSolution(lessthan30minutesbeforeuse):prepare20mLofStainingSolutionforeach8cm×10cmblotbyadding20μLof10mMNiCl2,20μLof1mg/mLbiotin-XNTA(fromStep2)and1-2μLof1mg/mLstreptavidin–alkalinephosphatase(madeinstep1.2)to20mLofBlockingBuffer.Mixwell.Oncethesolutionismade,itisreadytouserightaway,andremainsstableforatleast30minutes.

4)                  Incubatetheblotwith20mLofStainingSolution(fromStep3)atroomtemperaturefor30minutes.

5)                  Washtheblots,andpursuitforchemiluminescencedetection.

 

SampleProtocolforBiotin-XNTA–ProbedBlotStripping:

1)                  Ifdesired,theblotcanbestainedusingotherdetectionmethods.

2)                  Forstainingwithantibodiesorlectins,stripthebiotin-XNTAcomplexofftheblotbyincubatingitin62.5mMTris,0.2%SDS,50mMdithiothreitol(DTT),pH6.8,at50°Cfor40minuteswithgentleagitation.Orincubatedovernightin200mMacetateacidwith40mMEDTA,pH4.8.

3)                  WashtheblotinWashBufferatroomtemperature2-4timesfor5minuteseachandproceedwithantibodydetection.

References&Citations
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Alow-cost,high-performancesystemforfluorescencelateralflowassays
Authors:LindaGLee,EricSNordman,MartinDJohnson,MarkFOldham
Journal:Biosensors(2013):360--373

ImplementationofP22viralcapsidsasnanoplatforms
Authors:SebyungKang,MasakiUchida,AlisonO’Neil,RuiLi,PeterEPrevelige,TrevorDouglas
Journal:Biomacromolecules(2010):2804--2809

Calcium-dependentactivationoftransglutaminase2bynanosecondpulsedelectricfields
Authors:KeikoMorotomi-Yano,Ken-ichiYano
Journal:FEBSOpenBio


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