Difference gel electrophoresis

Abstract

Difference gel electrophoresis (DIGE) was invented to circumvent the inherent variability of 2-DE. This variability is a natural consequence of separating thousands of proteins over a large space, such as a 15 x 20 cm slab of polyacrylamide gel. The originators of 2-DE envisioned being able to compare cancerous cells and normal cells to understand what makes these cells different. Gel-to-gel variability made this an extremely difficult task. We reasoned that if both samples could be run on the same gel, then the inherent variability would be obviated. Thus, we created matched sets of fluorescent dyes that allows one to compare two or three protein samples on a single gel. In the 12 years since the description of DIGE first appeared in Electrophoresis, this founding paper has been cited over 660 times. This review highlights some of the improvements and applications of DIGE. We hope these examples are illustrative of what has been done and where the field is headed.

Proteomics unravels platelet function

“…Indeed, previous studies using the same assay did not demonstrate a difference in proteasome levels in CD138 ϩ cells from normal donors and myeloma patients. 6 More importantly, the data demonstrate inhibition of both proteasomes. Because the immunoproteasome is only expressed constitutively in hematopoietic cells, this may provide an explanation as to why Bcell malignancies are exquisitely sensitive to this class of therapeutics.…”

“…1,6 Classical 2D gel electrophoresis is hampered by intergel variability as well as difficulties in protein quantification. In 2D-DIGE, samples are labeled with different fluorescent dyes (typically Cy3 and Cy5, providing a wide dynamic range of fluorescence quantification) and run on the same 2D gel.…”

“…Thus, this provides a true reflection of the protein abundance throughout the investigated proteome. 6 Together with advances in the rapidly advancing fields of mass spectrometry and protein database content and management, 2D-DIGE is an ideal discovery tool for platelet research.…”

“…8 Amazingly, the use of proteasome-specific inhibitors demonstrates that the cellular processes controlled by each proteasome are in part distinct as selective inhibition of the constitutive proteasome does not affect IL-23 production in monocytes. 8 However, these selective inhibitors do not induce apoptosis when used individually, 6 thus overlapping functions are likely to exist as well. Therefore, normal and transformed B cells may be dependent on both proteasomes and explain why these cells are so sensitive to proteasome inhibition.…”

Proteomic analysis of oxidative stress-induced neuronal cell death by using two-dimensional fluorescence difference gel electrophoresis

“…Traditional twodimensional polyacrylamide gel electrophoresis (2D-PAGE) of cell lysates is the basis of proteomic studies, but there are several technical limitations (34). To overcome these technical limitations, we adopted the 2D DIGE technique, which is aimed at improving reproducibility and decreasing inter-gel variation (35). In addition, the subcellular fractionation method was applied to reduce sample complexity and investigate the translocation of proteins between subcellular compartments.…”