上海申鹿均质机有限公司
来自 : 蚂蚁淘
ClaireWalczakJanuary1996
Protocol:
Allstepsuptothedialysisatrt.
- PourcolumninTBS(=0.15MNaCl,20mMTrisClpH7.4).Weusea5mlcolumnfor25mlsserum.WashextensivelyinTBSafterprewashingasindicatedintheprotocolforcouplingpeptidetotheresin.
- Thawserum-dilute1:1withTBSandfilterthrougha0.2umfilter
- Loadtheserumoverthecolumn,takingatleast20minutetotal.
- Runthebreakthroughoverthecolumnfivetimes.AlternativelyyoucanuseaparastalticpumpandrecirculatetheserumONorjustbatchbindtheserumON.
- Washwith5colvolsTBS.
- Washwith10colvols0.5MNaCl,20mMTrisClpH7.4,0.2%Triton-X-100.
- Wash5colvolsTBS
- Elutewith0.15MNaCl,0.2MGlycine-HClpH2.0.Collect1mlfraction,witheachtubecontaining0.1mlof2MTrisClpH8.5
- WashwithTBSuntilpHisreequillibrated.
- Elutewith6MGuanidineHClinTBS,collecting1mlfractions.
- WashwithTBS+0.1%NaN3,andstoreat4degC.
- Todeterminewheretopoolfractions,spot1ulofeachfractionontonitrocellulosepaperandstainwithponceauS.Poolallfractionsthatshowpinkcolor.
- DialyzeONintoTBSoryourfavoritebuffer.
- Ifnecessarytheantibodycanbeconcentratedbysweatingthedialysisbags,orbyspin-concentrating.
- Bringtheazideconcentrationupto0.1%andstoreat4degCforuptothreemonths.Forlongerstoragefreezeinaliquotsandstoreat-80degCoraddglycerolto50%andstoreat-20degC.
Note:DonotpoolthelowpHandGuHCleluatesastheymayhavesignificantlydifferentproperties.WehavefoundthattheGuHClmayhavehigheraffinities,butalsomaycontainahigherfractionofpartiallydenaturedantibodythatcouldcontributetostainingbackground.Theproportionineachpoolvarieswiththepeptideimmunogen.
Thequalityoftheanti-peptideserumseemstoincreasewithmultipleboosts-thefirstbleedmaybefeeble.
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