ThemultiplecopiesoftheHAepitopepresentintheHATtagcanbedetectedbythemousemonoclonalantibodies12CA5(Boehringer)and16B12(MMS101R;BAbCO,Richmond,California)(seeTable1).OnWesternblotsagainstyeastprotein,theseantibodiesoftenrecognizeasinglecross-reactingbandofabout50or125kD,respectively,althoughthelevelofthe50kDpolypeptidecanbereducedbyusingculturesintheearlyphaseofexponentialdivision.Theantibodiesmayalsogivepunctatebackgroundwhenusedforimmunocytochemistry,althoughwehavefoundthatthiscanbeeliminatedbybothminimizingthespheroplastingprocessandpreadsorbingprimaryandsecondaryantibodiesinmultipleincubationsagainstfixedyeastcellssUSPendedinPBS.Arabbitpolyclonalantiseraisalsoavailable(101c500;BAbCO)butinourhandsthiswaslessreactive.

Theantibodycleanupprotocolisnowaseperatedocument

Protocol

1. Cellsaregrownin5mlsofYPADtoanO.D.600of0.75to1.
2. Tofix,1/10volumeof37%formaldehydeisaddedtotheculture,whichisshakenforafurther40minutesatroomtemperature.Cellsarethenrecoveredbycentrifugationat1400Xgfor2minutes,andwashed(i.e.gentlyresuspendedthenrecoveredandthesupernatantdiscarded)twicewithSolutionA(1.2Msorbitol,50mMKPO4,pH7).
3. Tospheroplast,cellsareresupendedin500ulofSolutionAcontaining0.1%b-mercaptoethanol,0.02%glusulaseand5ug/mlzymolyase.Thesuspensionisincubatedat37^oCwithoutshakingandcheckedperiodically.Assoonasthesettledcellpelletlosesitscreamy,yellowcolourandbecomestranslucent(30to45mins),cellsshouldberecoveredandwashedtwicewithSolutionA.
4. Cellsareresuspendedin200ulofsolutionA.Adropisplacedonapoly-L-lysinecoatedslideandallowedtositfor10minutes.Ifonedesirestoexamineseveraldifferentstrainsononeslide,multiwellslidesareavailablefromCarlsonScientific(Peotone,Illinois).
5. Excesssolutionisaspirated.TheadheredcellsarecoveredwithPBS(150mMNaCl,50mMNaPO4,pH7.4)plus0.1%BSA.Thesolutionisallowedtositfor5minutesbeforeaspiration.ThiswashisrepeatedtwicewithPBS/0.1%BSAcontaining0.1%NP40.
6. ThecellsarecoveredwithPBS/0.1%BSAcontainingtheprimaryantibody(Table1).Theslideissetonwetpapertowelsinasealedchamberandincubatedat4^oCovernight.
7. Excesssolutionisaspirated.ThecellsarecoveredwithPBS/0.1%BSA,whichisallowedtositfor5minutesbeforeaspiration.ThiswashisrepeatedwithPBS/0.1%BSAcontaining0.1%NP40,thenagainwithPBS/0.1%BSA.
8. ThecellsarecoveredwithPBS/0.1%BSAcontainingthesecondaryantibody(Table1).Theslideissetonwetpapertowelsinasealedchamberandincubatedatroomtemperaturefor2hours.
9. Excesssolutionisaspirated.ThecellsarecoveredwithPBS/0.1%BSA,whichisletsitfor5minutesbeforeaspiration.ThiswashisrepeatedwithPBS/0.1%BSAcontaining0.1%NP40,thenagainwithPBS/0.1%BSA.
10. Mountsolution(70%glycerolcontaining2%n-propylgallateand0.25ug/mlHoechst)isplacedonthecells.Acoverslipisplacedandsealedwithnailpolish.Slidesarestoredat-20^oCuntilexamination.

Antibodies

• mousemonoclonalanti-HA16B12(MMS101R,BAbCo,usedat1:200to1:1000dilution)
• mousemonoclonalanti-HA12CA5(12CA5,Boehringer,usedat1:200dilution)
• rabbitpolyclonalanti-HA(RS1010C,BAbCo,usedat1:200dilution)
• Cy3-conjugatedaffinity-purifiedgoatanti-mouseIgG(JacksonLabs;1mg/mlstockusedat1:300dilution)
• TexasRed-conjugatedaffinity-purifieddonkeyanti-rabbit(JacksonLabs;1mg/mlstockusedat1:200dilution)

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