Overview

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Ex/Em(nm)	None/None
MW	366.44
CAS#	N/A
Solvent	DMSO
Storage	F/D
Category	BiotinandItsDerivatives Thiol-ReactiveBiotins
Related	Thiol-ReactiveProbes BiochemicalAssays

Biotinandbiotinderivativesbindwithhighaffinitytobothavidinsandstreptavidins.BiotinanditsderivativescanbeconjugatedtomanybiomoleculeswithoutsignificantlyalteringtheBIOLOGicalactivityofthetargetmoleculessincebiotinisrelativelyasmallmolecule.Abiopolymer(suchasproteins)canreactwithseveralmoleculesofbiotinthat,inturn,caneachbindoneavidin.Thischaracteristicgreatlyincreasesthesensitivityofmanybiologicalassays.Biotinderivativesarewidelyusedforbiologicaldetectionsandpurification.Thisbiotinderivativeisusedtomodifythiolgroup.

Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

SampleProtocolforLabelingProteinswithBiotinC2Maleimide:

1)                  Dissolveyourthiol-containingproteinatconcentrationof1-10mg/mL(3-10mgistheoptimallabelingconcentration)usingPBSbuffer(20mM,pH7.2).

2)                  DissolvetheBiotinC2MaleimideinDMSOatconcentrationof5-10mg/mL.

3)                  MixtheBiotinC2Maleimide(fromStep2)andproteinsolution(fromStep1)at2:1molarratioofbiotin/protein,andshakethereactionmixtureatroomtemperaturefor2-4hours.

4)                  Filterthereactionmixturethroughaproteinspincolumnfor100µgto1mgproteinlabelingreaction;orpurifytheconjugateusinggelfiltrationonaproperlysizedSephadexG-25columnifthereactionscaleislargerthan1mg.

5)                  Collectthedesiredfractionsforyourimmediateuseorfreezedrythemforyourfutureuse.

 

SampleProtocolforLabelingSmallMoleculeswithBiotinC2Maleimide:

1)                  DissolveBiotinC2Maleimide(10-15mg/mL)andyourthiol-containingmoleculeinDMSOat1:1.2molarratioofbiotin/thiol-containmolecule.

2)                  Stirthereactionmixtureatroomtemperaturefor2-4hours.

3)                  PurifytheconjugateusingHPLC(ammoniumacetate/waterandacetonitrile,pH7.0).

4)                  Collectandpoolthedesiredfractions.

5)                  Combineandfreeze-drythepooledfractions.

References&Citations

CitationExplorer

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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