| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 500/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | ProteinBiochemistry Generalproteins |
| Related | BiochemicalAssays |
1.Prepare100XAvidinstocksolutionbyadding400µLddH2OintothevialofAvidin(ComponentA),Mixwell.
Note:Theunused100XAvidinstocksolutionshouldbedividedintosingleusealiquotsandstoredat-20oC
2.PrepareHABA/Avidinassaymixturebyadding200µLof100XAvidinstocksolution(fromStep1)into20
mLofHABAassaybuffer(ComponentB).Mixthereagentcompletely.
Note:TheunusedportionofHABA/Avidinassaymixturemightbestoredat4°Cuptooneweek.
3.QuantitateBiotinwitha96-wellMicroplate
3.1.Add20µLeachofbiotin-containingsamples,negativecontrol(ddH2Oorthesamebufferusedtodissolvebiotin-containingsample),andpositiveControl(ComponentC)intoa96-wellwhite/clearbottommicroplateasdescribedinTables1and2.
Table1Layoutofbiotin-containingtestsamples,negativeorpositivecontrolsinawhite/clearbottom96-wellmicroplate
NC | NC | TS | TS | …. | …. |
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PC | PC | …. | …. | …. | …. |
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TS | TS |
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…. | …. |
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Note:NC=NegativeControl,PC=PositiveControl,TS=TestSamples.
Table2Reagentcompositionforeachwell
PositiveControl | NegativeControl | TestSample* |
ComponentC:20μL | ddH2O:20μL | 20μL |
*Note1:Itisnecessarytotestthebiotin-containingsamplesatseveraldilutionstoensurethattheconcentrationofbiotiniswithintheassaylinearrange,2-16µMofbiotin(finalconcentration).
Note2:Avoidbufferscontainingpotassium,asitwillcauseprecipitationintheassay.
Note3:Freebiotinmustbeseparatedfromthebiotinylatedproteinbygelfiltrationordialysis.
3.2Add180µLofHABA/Avidinassaymixture(fromStep2)intoeachwellofthebiotin-containingsamples,negativecontrol,andpositivecontrol(seeStep3.1)tomakethetotalbiotinassayvolumeof200µL/well.
Note:ForaCuvetteFormat,add100μLsamplewith900μLHABA/Avidinassaymixture.
3.3Incubatethereactionmixtureatroomtemperaturefor5minutesbyshakingonaplateshakerat100-200rpm,protectedfromlightandavoidcreatingbubblesduringpipetting.
3.4 Monitortheabsorbancedecreasewithanabsorbanceplatereaderat500nm.
| References&Citations |  PrinterFriendlyVersion |
1. HensleyK.(2009)Detectionofproteincarbonylsbymeansofbiotinhydrazide-streptavidinaffinitymethods.MethodsMolBiol,536,457.
2. HoJA,HungCH.(2008)UsingLiposomalfluorescentbiolabelstodevelopanimmunoaffinitychromatographicbiosensingsystemforbiotin.AnalChem,80,6405.
3. LiebertK,JeltschA.(2008)DetectionandquantitationoftheactivityofDNAmethyltransferasesusingabiotin/avidinmicroplateassay.MethodsMolBiol,418,149.
4. MockDM,MockNI,LankfordGL,BurmeisterLF,StraussRG,WidnessJA.(2008)RedcellvolumecanbeaccuratelydeterminedinsheepusinganonrADIoactivebiotinlabel.PediatrRes,64,528.
5. MuratsuguM,YazawaA,FujiwaraS,NishidaS,FukuiT.(2008)Quantitationofbiotin-bindingimmunoglobulinsG,A,andMinHumanSeraUsingF(ab")2anti-humanimmunoglobulin-coatedmicroplates.BiolPharmBull,31,507.
6. BatchelorRH,SarkezA,CoxWG,JohnsonI.(2007)Fluorometricassayforquantitationofbiotincovalentlyattachedtoproteinsandnucleicacids.Biotechniques,43,503.
7. YazawaA,FukuokaK,HondaH,FukuiT,MuratsuguM.(2006)Biotin-proteinratiosandstABIlityofbiotinylatedimmunoglobulinsasstandardsforthequantitationofbiotin-bindingimmunoglobulins.BiolPharmBull,29,1480.
8. GuoLH,YangXQ.(2005)AnewchemicallyamplifiedelectRochemicalsystemforthedetectionofbiologicalaffinityreactions:directandcompetitivebiotinassay.Analyst,130,1027.
9. WuY,SimonsPC,LopezGP,SklarLA,BurandaT.(2005)Dynamicsoffluorescencedequenchingofostrich-quenchedfluoresceinbiotin:amultifunctionalquantitativeassayforbiotin.AnalBiochem,342,221.
10. BanksRE,CravenRA,HarndenPA,SelbyPJ.(2003)UseofasensitiveEnVision+-baseddetectionsystemforWesternblotting:avoidanceofstreptavidinbindingtoendogenousbiotinandbiotin-containingproteinsinkidneyandothertissues.Proteomics,3,558.
