E.Coli Westerns

Anne Spang spang at vms.biochem.mpg.de
Thu Dec 1 14:13:42 EST 1994


Hi Dan!
Have you ever tested the preimmune-serum, if the rabbit had already
antibodies against E.coli proteins? You could get rid of the background by
affinity purification. One possibility is to x-link your antigen to
CNBr-Sepharose ( or something equivalent) and put serum on the column.
Elution is usually done with glycin pH 2.5 and/or guanidinium
hydrochloride pH7.0.For teh exact protocol see Harlow and Lane "
Antibodies", a lab manual. Another possibilty might be the purification
via nitrocellulose strips on which you antigen is immobilized. The
advantage of this methode is, that you do not need pure antigen, because
you just cut out the band of your protein of interest. If you are
interested in this protocol,I can send it to you

Hope this helps!
Anne



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