Hi there, We also experienced the problem that certain antibodies loose activity when purifying via acid elution from protein A or G columns. In some cases, we found that a solution could be obtained by putting the hybridoma in Protein Free Hybridoma Medium (GibcoBRL; Cat N° 12040-051). Since there is no serum in this medium, the only protein should be the antibody. You can then "purify" the antibody by just concentrating the hybridoma culture supernate and dializing the concentrate against PBS. You have to check though whether you particular hybridoma will produce well in this medium. I hope this helps, Geert Raes Laboratory of Cellular Immunology Free University of Brussels Belgium Adrian Smith wrote: > Hi all, > We have been having some problems with some of our > monoclonals and would appreciate any advice or comments... > > Bascially we have two problems which may be related: after > purification on Protein A or G and elution (glycine pH2.5 then tris > pH8 neutralisation and dialysis against PBS), the antibody either > precipitates spontaneously at 4 degrees, or the biotinylated > conjugates rapidly lose activity (which may also be due to > precipitation - we haven't checked yet). We suspect low levels of > acid denaturation may cause ongoing spontaneous formation of > aggregates. Does anyone have any experience of this and how to get > around it (most mAbs are fine after purification this way and only > some mAbs seem to be affected)? > > Thanks, > Adrian > > -- > ****************************************************** > Adrian Smith (PhD Student) T CELL BIOLOGY GROUP > Centenary Institute of Cancer Medicine & Cell Biology > Locked Bag No.6 Newtown, NSW 2042 AUSTRALIA. > ******************************************************
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