Affinity purification and binding pH - (Sep/29/2006 )

Is PBS of pH = 9 too high for binding antibody to a peptide conjugated column? The peptide is the same as the immunogen. I am purifying rabbit serum, and in the past I have used up to 8.5 pH with good results, but this time I decided to try 9 and got very little yield. I know that it could be due to the rabbits and the immunogen, and there are other variables, but in general is the pH too high at all?

Well, if your peptide is charged and such a pH will signifiacntly change the overall charge property. I think this pH may be close to some elution conditions for immunoaffinity separation. Is there a particular reason to use it?

Not really. I just read for protein A and G purifications that a higher binding pH usually helps more antibody bind the column, but this may be different for affinity columns. I will go back to a pH between 7.4 and 8 in the future. I found out that the column had been used for a few purifications before I got hold of it, and that they got low yields as well. So, just to be safe I won't use such a high pH, but that probably wasn't the reason I got a low yield. It was probably just a low immunogenic peptide to begin with.

I dont think that protein A and G bind to the antigen binding sites.

I agree perhaps there maybe other reasons for the low binding capacity.

check the isoelectric point for the peptide..