Abstract
Two-dimensional difference gel electrophoresis (2D DIGE) is a modified form of 2D electrophoresis (2D E) that allows one to compare two or three protein samples simultaneously on the same gel. The proteins in each sample are covalently tagged with different color fluorescent dyes that are designed to have no effect on the relative migration of proteins during electrophoresis. Proteins that are common to the samples appear as “spots” with a fixed ratio of fluorescent signals, whereas proteins that differ between the samples have different fluorescence ratios. With conventional imaging systems, DIGE is capable of reliably detecting as little as 0.2 fmol of protein, and protein differences down to ± 15%, over a ~10,000-fold protein concentration range. DIGE combined with digital image analysis therefore greatly improves the statistical assessment of proteome variation. Here we describe a protocol for conducting DIGE experiments, which takes 2–3 days to complete. We have further improved upon 2D DIGE by introducing in-gel equilibration to improve protein retention during transfer between the first and second dimensions of electrophoresis and by developing a fluorescent gel imaging system with a millionfold dynamic range.
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Acknowledgments
The development and optimization of this DIGE protocol would not have been possible without the efforts and dedication of my students and staff: Mustafa Ünlü, Liz Morgan, Chris Lacenere, Surya Viswanathan, Lei Gong, Mamta Puri, Susan Dowd, and Amber Lucas, as well as current and past members of the undergraduate proteomics platoon.
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Blundon, M., Ganesan, V., Redler, B., Van, P.T., Minden, J.S. (2019). Two-Dimensional Difference Gel Electrophoresis. In: Kurien, B., Scofield, R. (eds) Electrophoretic Separation of Proteins. Methods in Molecular Biology, vol 1855. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8793-1_20
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DOI: https://doi.org/10.1007/978-1-4939-8793-1_20
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