MMUNOSCREENING OF WITH ANTIBODY PROBES

AdsroptionofAnti-E.coliAntibodies

GrowE.colistrainY1090inLbrothat37°Covernight.Transferto10mMMgSO4andstoreat4°Cforfutureuse.Thesecellsaregoodforabout1week.

Soakone132mmnitrocellulosemembraneperPetridishin10mMIPTG(MW=238.31).Brieflyblottheexcesssolutionfromthefilters,andwrapthemincellophane.Theseshouldbemoistandstoredat4°C.

Wetypicallyadsorbserumwithfive150mmPetridishesoflysedE.colitoreduceanti-E.coliantibodybackground.Theseantibodiesarepresentinallhumansera,rabbitsera,androdentsera.ForeachPetridishcontainingsolidifiedLBagar,mix0.6mloftheY1090overnight-saturatedculturewith30,000pfusoflamBDa-gt11(dilutedinTMG)inglasstesttubesandincubateat37-42°Cfor15minuteswithoutshaking.

Platethismixturewith7-7.5mltopagarper150mmPetridish.Letthedishessitonthelabbenchforabout5minutestosolidifythetopagar.Thentransferthemtoa42°Cincubator.Checkforplaqueformationafter3hours.Ifplaquesareseen,gotostep5afteranother30minutes(i.e.atotalincubationtotaltimeof3.5hours).Ifplaquesarenotseenorareverysmall,waitanotherhourbeforecontinuing.Ifnoplaquesareseenat4hours,youprobablyscrewedupthedilutionandmightaswellstartover!

CarefullylayerasingleIPTG-soakedmembraneoneachPetridish(avoidtrappingairbubblesbetweenthemembraneandtheagar),andtransferthedishestoa37°Cincubatorfor3hours.

Flipmembranesupsidedownonthedishesandincubateforanother2-3hours.

RemovemembranesfromplatesandwashfourtimesinTBSTfor5minuteseachatroomtemperature.

Blockmembraneswithgentleagitationfor1hourina5%non-fatdrymilksolutionmadeinTBST(orblockovernightat4°C).

WashmembranesfourtimesinTBSTfor5minuteseachatroomtemperature.

Diluteserum1:100in3%BSAand0.02%sodiumazideinTBST

Transferdilutedserumina150mmPetridish.Placeonemembraneintheserumfor1houratroomtemperature,shakinggently.For30mlofdilutedserum,weusuallyadsorb5membranessequentially.Althoughthebackgroundmightstillbehigh(E.colititersvarygreatlybetweeninpiduals),weprefertodolibraryscreeningsusingpartiallyvs.totallyadsorbedserum.Withsuchsera,eachplaqueisobserved,andpositiveplaquesstandoutincolorintensityagainstabackgroundofnegativeplaques.(Note:Iftheserumisveryweak,additionaladsorptionsmaybenecessary.)KeepasmallsectionofeachmembraneinTBSTforevaluationafteradsorptions.Numbereachofthesesectionswithpencilindicatingtheorderofadsorption.

WashthesmallmembranesectionsinTBSTfor5minutes,4times,atroomtemperature.

Incubatethesemembranesectionswithsecondaryantiserumfor1houratroomtemperature(Fordetectinghumanantibodies,weuseSigma#A-3150:goatanti-humanIgGalkalinephosphataseconjugate,diluted1:1000)

WashmembranesinTBSTfor5minutes,4timesatroomtemperature.

Developmembranesbydiluting33µlNBTand66µlBCIPin10mlofalkalinephosphatasebuffer.Soakmembranesectionsinthissolutionfor5-10minuteswhilemixing.Keepthismixtureunderfoiltoreduceexposuretolight.