Antibody cleanup-蚂蚁淘生物

Inordertodecreasetheamountofnonspecificstaining,itisoftennecessarytopreabsorbprimaryandsecondaryantibodiestoyeastcellslackingtheantigenpriortouse.A1:1mixtureoffixedyeastwholecellsandspheroplastsareusedforthispurpose.

Growcellsin200mlsofYPDat30^oCtoanOD600of1.

Addformaldehydetothe200mlculturetoafinalconcentrationof3.7%.Fixfor1houratroomtemperaturewithshaking.

Centrifugecellsat3000Xg.ResUSPendin200mlsSolutionA.Repeatwash.

Pelletcellsasaboveandresuspendin4mlsSolutionA.Reserve2mlsofthecellsuspension.

Spheroplasttheremaining2mlofthecellsuspensionbyadditionofbeta-mercaptoethanolto0.1%,glusulaseto0.05%,andzymolyase(100T)to60ug/ml.Incubate1hourat37^oC.

Pelletspheroplastsasabove.Resuspendin10mlsSolutionAandpelletagain.

Combinethesuspensionofwholecellswiththespheroplastsandpelletasabove.Resuspendin10mlsofPBS.

Put200ul(3mg/ml)oftheantibodytoanEppendorftube.Add0.8mlsofPBS.

Pellet1mlofthecellsuspensionanddiscardthePBSsupernatant.Addtheantibodysolutiontothecellpellet,gentlyresuspendthecells,andincubatefor1houronice.

Pelletcells,givinganantibodysupernatant.Addthistoacellpelletpreparedasinstep9.Beverycarefulnottogetsupernatantsmixedup!

Repeatsteps9+10seventimes,orasoftenasnecessarytodecreasethebackground.

Pelletcells.Saveantibodysupernatant,whichisnowdiluted1:5fromitsinitialconcentration.Theseantibodiescanbestoredat4^oCforseveralweeksorat-70^oCforlongerperiods.

Solutions

SolutionA	1.2Msorbitol,50mMKPO4,pH7.0
PBS	150mMNaCl,50mMNaPO4,pH7.4

Solutions

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