2D-DIGE analysis of eye lens proteins as a measure of cataract formation

Abstract

This chapter describes the basics of two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of two distinct proteomes. The example given describes the analysis of male and female rat lens soluble proteins labeled with fluorescent Cy3 and Cy5 dyes in comparison to a pooled standard labeled with Cy2. After labeling the proteomes are mixed together and electrophoresed on the same 2D gels. Scanning the gels at wavelengths specific for each dye allows direct overlay the two different proteomes. Differences in abundance of specific protein spots can be determined through comparison to the pooled standard.