Protocol:

Allstepsuptothedialysisatrt.

1. PourcolumninTBS(=0.15MNaCl,20mMTrisClpH7.4).Weusea5mlcolumnfor25mlsserum.WashextensivelyinTBSafterprewashingasindicatedintheprotocolforcouplingpeptidetotheresin.
2. Thawserum-dilute1:1withTBSandfilterthrougha0.2umfilter
3. Loadtheserumoverthecolumn,takingatleast20minutetotal.
4. Runthebreakthroughoverthecolumnfivetimes.AlternativelyyoucanuseaparastalticpumpandrecirculatetheserumONorjustbatchbindtheserumON.
5. Washwith5colvolsTBS.
6. Washwith10colvols0.5MNaCl,20mMTrisClpH7.4,0.2%Triton-X-100.
7. Wash5colvolsTBS
8. Elutewith0.15MNaCl,0.2MGlycine-HClpH2.0.Collect1mlfraction,witheachtubecontaining0.1mlof2MTrisClpH8.5
9. WashwithTBSuntilpHisreequillibrated.
10. Elutewith6MGuanidineHClinTBS,collecting1mlfractions.
11. WashwithTBS+0.1%NaN3,andstoreat4degC.
12. Todeterminewheretopoolfractions,spot1ulofeachfractionontonitrocellulosepaperandstainwithponceauS.Poolallfractionsthatshowpinkcolor.
13. DialyzeONintoTBSoryourfavoritebuffer.
14. Ifnecessarytheantibodycanbeconcentratedbysweatingthedialysisbags,orbyspin-concentrating.
15. Bringtheazideconcentrationupto0.1%andstoreat4degCforuptothreemonths.Forlongerstoragefreezeinaliquotsandstoreat-80degCoraddglycerolto50%andstoreat-20degC.

Note:DonotpoolthelowpHandGuHCleluatesastheymayhavesignificantlydifferentproperties.WehavefoundthattheGuHClmayhavehigheraffinities,butalsomaycontainahigherfractionofpartiallydenaturedantibodythatcouldcontributetostainingbackground.Theproportionineachpoolvarieswiththepeptideimmunogen.

Thequalityoftheanti-peptideserumseemstoincreasewithmultipleboosts-thefirstbleedmaybefeeble.

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