Antibody Affinity Purification - peptide column (Jun/30/2011 )
we ordered a antibody and recieved the anti-serum. Now I did the affinity purification with the peptide column. After washing the column with PBS pH 7,4 I eluted the antibody with pH 2,7 Glycine in Eppendorf cup containing 50µl of Tris pH 9. When I measured the fractions with Nanodrop, I prior blanked with Glycine+Tris...The values are really messed up and jumping high-low without any sense. Does anyone has an idea why? Should I use convenient Spectrophotometer with Lowry or Bradfort rather than Nanodrop? Any suggestions are appreciated.
Edit: I ask myself...Should I do size exclusion column directly after affinty purification, somehow I see a small pellet in some fractions...perhaps tris and glycine are precipitating my antibody. Desalting and changing the buffer to PBS should do the trick maybe...then try nanodrop
If there were a lot of antibodies in your antiserum, then precipitation is quite likely. That would also explain why the OD measurements were all over the place. I would definitely dialize the eluted fractions, however I wouldn't apply the pellet to a SEC column as it might stop the flow. Use a dialysis membrane instead. Some Abs have a pI close to 7 so they precipitate in PBS as well. In that case a buffer with a lower pH is better.
Thank you for your advise.