1. FilterSup.in1LCostarFilterusingpre-filter.
2. Add50mMTrispH7.8(50mlof1Mstock/Liter).100ml
3. Add40gammoniumsulfateforevery100mlSup.(slowly).800g
4. StirO/Nincoldroom.
5. Pourintoplasticbottles.Spinat4^oC,7,500rpmfor20mininJA-10rotor.
6. Prepare4L100mMTris/pH7.8(400mlof1Mstock/4L)orBindingbuffer.
7. DiscardSup.ResUSPendpelletsin10mlof100mMTrisbufferorBindingbuffer.Andpoolinto50mlFalcontubes(trynottomakebubbles).
8. Use3.2ml/cm12-14,000MWdialysistubing.
   • Heattubingin500mlH₂Oinmicrowave.Notboiling.
   • RinsetubinginfreshH₂Oseveraltimes.
   • Testeachtubew/H₂Oanddiscard.
9. Addproteinmixturetodialysistube.Stirslowlyin100mMTrisbufferorBindingbufferuntilpinkcolorisout.
10. Transferproteinmixture(Ab)to50mlFalcontubestodeterminevolume.
11. Dilutethemixture1:1withBindingbuffer.
12. Filterthrough0.45umfilters(useprefilters).

1. PrepareProteinAcolumn.
2. Runbindingbuffer(pH9.0)~200ml.
3. Filterproteinmixture
4. AddproteinmixtureorculturesupernatantcontainingAb(adjustpHto7.8withBindingbuffer;redcolor)totheProteinAcolumn.

   MouseantibodiesoftheIgG1subclassdonothaveahighaffinityforproteinA.PurificationonproteinAbeadsusingstandardconditionswillyieldapproximately1/10theamountofantibodycomparedwithothersubclasses.IncaseofIgG1subclass,add3.3MNaCl(192.85g/L)tocrudeantibodypreparation(serum,tissueculturesupernatant,orascites).

5. ApplyBindingbufferagain~200ml.
6. ApplyElutionbuffer~100ml.
7. Collect3mlfractionsin5mltubeswith700ul1MTrispH9.0alreadyinbottomoftubetoneutralize(collectat~5min/fraction).
8. 25tubesaresufficientforcollection.Ingeneral,youcanseehighAbconcentrationsin7-8tubes.
9. Test1ulonpHpaper.
10. ReadODtoknowAbconcentrations.
11. OD(Absorbanceat280nm)/1.35=Xmg/ml.
12. StoreAbsat-20^oCorfurtherconcentratebyusingCentriprep.Andstoreat-20^oC.
13. RegenerateAbcolumnwithRegeneratebuffer(~200ml).

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