Preparing Colloidal Gold for Electron Microscopy

Colloidalgoldhasbeenusedforcenturiesinthepreparationofstainedglassforwindowsandfineglassware.Inrecentyears,colloidalgoldparticleshavebecomeausefultoolformicroscopists.ColloidalgoldparticlesareespeciallyusefulforBIOLOGicalelectronmicroscopy.Someofthereasonswhyarelistedbelow.

• Homogeneouspreparationsofparticlesvaryinginsizefrom3nmto20nmcanbeeasilyprepared.
• ColloidalgoldsUSPensionsareinexpensivetoprepare.
• Mostproteinscanbeeasilycoupledtocolloidalgoldparticles.
• Proteinscoupledtogoldparticlesdonotappeartolosetheirbiologicalactivity.
• Thecolloidalgoldparticlescanbeeasilyseenintheelectronmicroscope.
• Colloidalgoldprobescanbeusedforlightmicroscopy.Thelargergoldparticlescanbedirectlyobservedbythelightmicroscope.SmallerparticlesaredetectedbysilverenhancementorepipolarizedIllumination.
• ThesameprobescanbeusedforbothLMandTEMimmunocytochemistry.

Thispagewillexplainasimple,publishedprotocolforpreparingcolloidalgoldparticles,howtocoupletheseparticlestoproteinAandhowtopurifytheprobesaftertheyhavebeenmade.

Tomake100mlofgoldsol,twostocksolutionshavetobeprepared.

SolutionA

80mldistilledwaterand1ml1%aqueousgoldchloride.

SolutionB

4ml1%tri-sodiumcitrate.2H2O+16mlH2O+variableamountof1%tannicacid[Mallinckrodt#1764](seetable1below).

When1mlormoretannicacidisneeded,addanequalamountof25mMpotassiumcarbonateforpHadjustment.

WarmupsolutionsAandBto60°Candmixthemwhilestirring.Whentheredcolorhasformedheatupto95°Candcoolthesolutiononice.Thelargerparticles(wherelowerconcentrationsoftannicacidareused)takelongertoformandtheredcolorcantakeupto1hrtodevelop.

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AgoldsolwillbindproteinsmoreefficientlywhenthepHofthesolutionisclosetothepIoftheprotein(forproteinAthepIispH5.1).AtighterbindingoccursathigherpHbutthismayhaveadenaturingeffectontheproteinmakingtheprobelesseffective.Havingtoomuchproteincoupledtothegoldparticlesmayalsobedisadvantageous:someoftheweaklyboundproteinmaydetachfromtheparticles.Thiswillmaketheprobelesseffectivebecausethefreeproteinwillcompeteforbindingsiteswiththegold-labelledprotein.HorrisbergerandClerc(1985,LabellingofcolloidalgoldwithproteinA.Aquantitativestudy.Histochemistry,82,219-223)recommendbindingtheproteinAtocolloidalgoldatpH6.0.

CheckthepHofthegoldsolwithpHpaper(thegoldsolwillblockapHelectrode)andadjustthepHwith0.1Nsodiumhydroxide.

ProteinA(BoehringerMannheim)isdissolvedindistilledwaterat1mg/ml.AmicrotitrationassaywillshowthecorrectamountofproteinAtoaddtothegoldsol(between4-6µg/ml).

AddtheproteinAwhilestirringthegoldsol.After5minadd10%bovineserumalbumin(BSA)inPBStoafinalconcentrationof0.2%(2ml/100ml)tomaximallystABIlizethesol.Returntotopofpage

TheproteinA-goldiscentrifugedinaTi70rotorinaBeckmanultracentrifugeattheappropriatespeed(seetable2)for30minat4°C.Atthecorrectspeedthegoldparticleswillsettletothebottomofthetubeasaloosepellet.Removethesupernatantwithoutdisturbingthepelletandre-suspendtheloosepartofthepelletinPBScontaining0.2%BSA.

AsecondcentrifugationstepdownagrADIentwillremoveanygoldparticlesofthewrongsize.Thisisdoneona10-30%continuousglycerolgradientat4°C.Layer1-2mlofproteinAgoldontothetopofthegradient,spininanSW40rotorattheappropriatespeed(seetable3)for45min.at4°C.Thedarkredbandinthemiddleofthegradientiscollected.AllaggregatedproteinAgoldparticleswillhavebeenremoved.Returntotopofpage

Immunogoldprobesmayloseactivitywithinweeks,duetothedissociationoftheproteinsfromthegoldparticles.

DialysetheproteinAgoldagainst50%glycerolinPBSandstoreat-20°Corfreezedownsmallaliquotsinliquidnitrogenandstoreat-70°C.

TodeterminetheconcentrationatwhichtousetheproteinAgold,measuretheopticaldensity(OD)at520nmofa1:100dilutedsolutioninPBS.UseadilutionwithanO.D.ofbetween0.05and0.1wherethereisnosignificantbackground.Ifusingaprimaryantibodythentheoptimaldilutionofthisfirstantibodymustbeknown.SectionscanbetreatedwithproteinAgoldalonetodeterminethebackgroundlabelling.Returntotopofpage

Table1:

Theinfluenceofthetannicacidconcentration,duringgoldsolformation,onthesizeofthegoldparticles.

When1mlormoreoftannicacidisusedanequalamountof25mMpotassiumcarbonatemustbeaddedtoneutralizesolutionB.Returntotopofpage

Table2:

Firstcentrifugation,usingaTi70rotorfor30minat4°C.Thisstepwillconcentratethecolloidalgoldprobeinthebottomofthetube.

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Table3:

SecondcentrifugationusingSW40rotorfor45minat4°C.Thiswillseparatethedifferentsizesofgoldparticlealongthegradient.

The4nmgoldmustbecentrifugedfor1hrforbestresults.The5nmgoldcanalsobecentrifugedat35000rpmfor1hr.

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BSA-goldisausefulMarkerforstudyingtheendocyticprocessesinmammaliancells.Typically,thelivingcellsareincubatedinaBSA-goldsuspensionwithafinalOD,at520nm,of5,meaningthatalargeamountofgoldprobeisneededfortheseexperiments.TheaboveprotocolforpreparingproteinA-goldcanbefollowedforpreparingBSA-goldbutBSAissubstitutedfortheproteinA(weuse2.4µg/ml).Thecentrifugationvaluesarethesame.