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
Edited by Danio ivanio, 30 June 2011 - 07:34 AM.