I am going to start DIGE analysis. When I coloured my protein extract with ATTO DYE according manual there is recommendation to separate unconjugate dye from proteins by using Sephadex G-25 column. I had 0.2 ml of coloured proteins with Atto dye 488 and when I put them on the column, I take 1 ml of elution solution with proteins, so my sample was diluted a lot. And when i use Atto 635, all column turn to blue and it was impossible to take the fraction of proteins. Can You give me advice what to do? Should I separate two samples coloured with ATTO dyes together on one Sephadex column or should I use one column per one sample and per one dye.