Microspheresshouldbeprotectedfromprolongedexposuretolightthroughoutthisprocedure.

1.ResUSPendthestockuncoupledmicrospheresaccordingtotheinstructionsdescribedintheProductInformationSheetprovidedwithyourmicrospheres.

2.Transfer5.0x10⁶ofthestockmicrospherestoaUsascientificmicrocentrifugetube.

3.Pelletthestockmicrospheresbymicrocentrifugationat≥8000xgfor1-2minutes.

4.Removethesupernatantandresuspendthepelletedmicrospheresin100μLdH₂Obyvortexandsonicationforapproximately20seconds.

5.Pelletthemicrospheresbymicrocentrifugationat≥8000xgfor1-2minutes.

6.Removethesupernatantandresuspendthewashedmicrospheresin80μL100mMMonobasicSodiumPhosphate,pH6.2byvortexandsonicationforapproximately20seconds.

7.Add10μLof50mg/mLSulfo-NHS(dilutedindH20)tothemicrospheresandmixgentlybyvortex.

8.Add10μLof50mg/mLEDC(dilutedindH₂0)tothemicrospheresandmixgentlybyvortex.

9.Incubatefor20minutesatroomtemperaturewithgentlemixingbyvortexat10minuteintervals.

10.Pellettheactivatedmicrospheresbymicrocentrifugationat≥8000xgfor1-2minutes.

11.Removethesupernatantandresuspendthemicrospheresin250μLof50mMMES,pH5.0byvortexandsonicationforapproximately20seconds.SeeTechnicalNote1.

12.Pelletthemicrospheresbymicrocentrifugationat≥8000xgfor1-2minutes.

13.Repeatsteps11.and12.Thisisatotaloftwowasheswith50mMMES,pH5.0.

14.Removethesupernatantandresuspendtheactivatedandwashedmicrospheresin100μLof50mMMES,pH5.0byvortexandsonicationforapproximately20seconds.

15.Add125,25,5or1μgproteintotheresuspendedmicrospheres.(Note:Werecommendtitrationinthe1to125μgrangetodeterminetheoptimalamountofproteinperspecificcouplingreaction.)

16.Bringtotalvolumeto500μLwith50mMMES,pH5.0.

17.Mixcouplingreactionbyvortex.

18.Incubatefor2hourswithmixing(byrotation)atroomtemperature.

19.Pelletthecoupledmicrospheresbymicrocentrifugationat≥8000xgfor1-2minutes.

20.Removethesupernatantandresuspendthepelletedmicrospheresin500μLofPBS-TBNbyvortexandsonicationforapproximately20seconds.SeeTechnicalNote2.

21.Incubatefor30minuteswithmixing(byrotation)atroomtemperature.(Note:Optional–performthisstepwhenusingthemicrospheresthesameday.)

22.Pelletthecoupledmicrospheresbymicrocentrifugationat≥8000xgfor1-2minutes.

23.Removethesupernatantandresuspendthemicrospheresin1mLofPBS-TBNbyvortexandsonicationforapproximately20seconds.SeeTechnicalNote3.

24.Pelletthemicrospheresbymicrocentrifugationat≥8000xgfor1-2minutes.

25.Repeatsteps23.and24.Thisisatotaloftwowasheswith1mLPBS-TBN.

26.Removethesupernatantandresuspendthecoupledandwashedmicrospheresin250-1000μLofPBS-TBN.

27.Countthemicrospheresuspensionbyhemacytometer.Calculation:Totalmicrospheres=count(1cornerof4x4section)x(1x10⁴)x(dilutionfactor)x(resuspensionvolumeinmL)

28.Storecoupledmicrospheresrefrigeratedat2-8°Cinthedark.

TechnicalNote1:Couplingcanbeperformedin100mMMES,pH6.0withsimilarresults.Forsomeproteins,bettersolubilityandbettercouplingmaybeachievedatahighercouplingpHorinADIfferentbuffer.Ifyourproteindoesnotcouplesatisfactorilyundertheserecommendations,tryPBS,pH7.4asanalternatecouplingbuffer.

TechnicalNote2:EitherPBS-TBN(PBS,0.1%BSA,0.02%Tween-20,0.05%Azide,pH7.4)orPBS-BN(PBS,1%BSA,0.05%Azide,pH7.4)maybeusedasBlocking/StorageBuffer.

TechnicalNote3:EitherPBS-TBNorPBS,0.05%Tween-20maybeusedasWashBuffer.