I recommend doing a subtractive purification only. Remove the undesired reactivity of each antibody by putting it over a column containing the epitope peptide of the other antibody. The purified antibody will not bind to the column and will come through. The reason I think you should not attempt to purify each antibody by binding it to its own epitope is that it will be difficult to elute the antibodies with the highest affinities and you could lose them.

I'm guessing the immobilized albumin agarose was used because the epitope peptide was conjugated to albumin to make the antigen, with the result that the antiserum contains anti-albumin antibodies. If your antisera used keyhole limpet hemocyanin (KLH) conjugation, you probably can leave those antibodies in the antiserum, as there should not be any KLH in your tissue samples to cross-react.

If albumin was used, you can make an anti-albumin column by binding the same type of albumin to cyanogen bromide-activated Sepharose, which is commercially available.

Hello Maria,

I think you should check immunoglobulin types of your Antibodies first. After that, you can chose an affinity resin accordingly. Because, protein A and Protein G resins do not select all types of immunoglobulins with same efficiency.

I am also agree with Adam for using specific epitope bond sepharose. If you have enough antigen, you can try it first. If not, you can use prepacked affinity colums from Biorad or GE.

I used GE prepacked colums for purifying my antibody, but it did not solve the unspecific binding problem of ours. I hope it solves yours. 

Did you try competition asssay to understand your antibodies specificity against your target proteins by western blot or Eliza?

Thank you very much, Adam and Cigdem.  My antisera used KLH conjugation.  There is no albumin in my antigen.  I think the immobilized albumin was used as a first step to remove from plasma substances which bind to albumin.  I am using  the rabbit bleeds to purify my antibodies, so, I guess I need to clean it from complement proteins, etc. in order to not overwhelm my column with the peptide coupled, right?.  That is why I was thinking about the Affi-gel blue column as a first step. I think you are right about the third column, Adam.  I guess they used it to get a better purified antibody.

I think the competition studies were done, and there was no problem in the western blot.  I will re-check that.  I just got incorporated to this project.

Thanks again to both of you.

Purification of the immunoglobulin away from other proteins in the antiserum is not necessary for Western blots, immunohistochemistry, or pull-down experiments. It does not improve the specificity of the antiserum. So you do not need protein A or G columns.

Thanks a lot, Adam.

Interesting. I was wondering why the two antisera to the two peptides cross-react. Is it because of the KLH carrier? or are there shared structural motifs in the two peptides?

Hi Bruno,

I am doing IHC in vertebrate tissues, so, KLH should not cause cross-reaction. I do not know if the peptides or other proteins in the tissue share epitopes...

Muchas gracias,

Maria B.