Inordertodecreasetheamountofnonspecificstaining,itisoftennecessarytopreabsorbprimaryandsecondaryantibodiestoyeastcellslackingtheantigenpriortouse.A1:1mixtureoffixedyeastwholecellsandspheroplastsareusedforthispurpose.

1. Growcellsin200mlsofYPDat30^oCtoanOD600of1.
2. Addformaldehydetothe200mlculturetoafinalconcentrationof3.7%.Fixfor1houratroomtemperaturewithshaking.
3. Centrifugecellsat3000Xg.ResUSPendin200mlsSolutionA.Repeatwash.
4. Pelletcellsasaboveandresuspendin4mlsSolutionA.Reserve2mlsofthecellsuspension.
5. Spheroplasttheremaining2mlofthecellsuspensionbyadditionofbeta-mercaptoethanolto0.1%,glusulaseto0.05%,andzymolyase(100T)to60ug/ml.Incubate1hourat37^oC.
6. Pelletspheroplastsasabove.Resuspendin10mlsSolutionAandpelletagain.
7. Combinethesuspensionofwholecellswiththespheroplastsandpelletasabove.Resuspendin10mlsofPBS.
8. Put200ul(3mg/ml)oftheantibodytoanEppendorftube.Add0.8mlsofPBS.
9. Pellet1mlofthecellsuspensionanddiscardthePBSsupernatant.Addtheantibodysolutiontothecellpellet,gentlyresuspendthecells,andincubatefor1houronice.
10. Pelletcells,givinganantibodysupernatant.Addthistoacellpelletpreparedasinstep9.Beverycarefulnottogetsupernatantsmixedup!
11. Repeatsteps9+10seventimes,orasoftenasnecessarytodecreasethebackground.
12. Pelletcells.Saveantibodysupernatant,whichisnowdiluted1:5fromitsinitialconcentration.Theseantibodiescanbestoredat4^oCforseveralweeksorat-70^oCforlongerperiods.

Solutions

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