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1、Activation1. Washparticles(e.g.,100mgof1pmcarboxylatedlatexbeads)intocouplingbuffer(50mMMES,pH6.0).Suspendin5mlcouplingbuffer.Theadditionofadilutedetergentsolutionmaybedonetoincreasebeadstabilityandpreventclumping(e.g.,0.01percentSDS).Avoidtheadditionofanycomponentscontainingcarboxylatesoramines(suc
2、hasacetate,glycine,Tris,imidazole,etc.).Also,avoidthepresenceofthiols(e.g.,DTT,2-mercaptoethanol,etc.),asthesewillreactwithEDCandeffectivelyinactivateit.2. Add100mgofEDCand100mgofsulfo-NHS.Mixtodissolve.Tofacilitatefasterdissolution,EDCandsulfo-NHSmaybedissolvedimmediatelybeforeuseasaconcentratedsto
3、cksolutioninreactionbufferandthenanaliquotofthissolutionaddedtotheparticlesuspensiontoobtainthecorrectfinalconcentration.3. Reactfor15minutesatroomtemperature.4. Quicklywashbeads2timeswithcouplingbufferusingcentrifugationandresuspendusingasonicprobein5mlofthesamebuffer.Coupling5. Dissolveproteintobe
4、coupledin5mlcouplingbufferataconcentrationsufficienttoprovide1-to10-foldmolarexcessofligandoverthemaximalcalculatedmonolayerconcentrationfortheamountandtypeofbeadsused.Forparticlemanufacturersreportingacarboxylateconcentrationinmeq/g,thisisequivalenttopmol/mg.Theoptimalproteinconcentrationshouldbeop
5、timized.Note:Toolowaproteinconcentrationmayresultinparticlecrosslinking.Forcouplingofexpensiveantibodiesthatmaynotbeavailableinenoughquantitytoreachtheoptimalmolarratioontheparticles,theadditionofanotherprotein(i.e.,bovinegammaglobulinorBSA)maybedonetotakeupremainingreactivesites.6. Combinetheprotei
6、nsolutionwithparticlesandmixthoroughly.7. Reactatroomtemperature2-4hourswithmixing.8. Washbeadswithcouplingbufferandresuspendinthesamebuffercontaining100mMofanamine-containinghydrophilicquenchingmoleculetoblockexcessreactivesites(i.e.,ethanolamineorTris).9. Washbeadsandresuspendinanappropriatebufferforstorage.
