Protocol abstract

Nature Protocols 1, 1732 - 1742 (2006)
Published online: 16 November 2006 | doi:10.1038/nprot.2006.256

Subject Categories: Biochemistry and protein analysis | Genomics and proteomics | Isolation, purification and separation

Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling

Nilesh S Tannu1 & Scott E Hemby1

Quantitative proteomics is the workhorse of the modern proteomics initiative. The gel-based and MuDPIT approaches have facilitated vital advances in the measurement of protein expression alterations in normal and disease phenotypic states. The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the multiplexing fluorescent two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). 2D-DIGE is based on direct labeling of lysine groups on proteins with cyanine CyDye DIGE Fluor minimal dyes before isoelectric focusing, enabling the labeling of 2–3 samples with different dyes and electrophoresis of all the samples on the same 2D gel. This capability minimizes spot pattern variability and the number of gels in an experiment while providing simple, accurate and reproducible spot matching. This protocol can be completed in 3–5 weeks depending on the sample size of the experiment and the level of expertise of the investigator.

  1. Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.

Correspondence to: Scott E Hemby1 e-mail:


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