DIGE Differential (fluorescence) 2D gel electrophoresis
DIGE utilizes mass- and charge-matched, spectrally resolvable fluorescent dyes (Cy2, Cy3 and Cy5) to label up to 3 different protein samples (or 2 samples and 1 pooled internal standard) in vitro prior to 2-D electrophoresis. Both the control and the experimental sample are run in a single polyacrylamide gel but imaged separately (Typhoon 9410) at a discrete wavelength for each respective dye. Hence, the images are overlaid without any ‘warping’ which substantially raises the confidence with which protein changes between samples can be detected and quantified.
- Typically, differential expression ratios are obtained on 1,000 or more protein spots.
- DIGE provides a top-down view into changes in the post translational proteome.
- Protein spots are automatically selected via user-defined criterion (e.g., differential expression between a control versus cancer sample of >1.3-fold) and subjected to robotic spot excision, trypsin digestion, and MALDI-Tof/Tof (AB Model 4800)
- Protein identification is based on highly accurate masses and MS/MS sequence data from multiple tryptic peptides (e.g., 5-10 peptides/protein is common)
- Very hydrophobic, basic, or very large or small molecular weight proteins (> 200K or < 5K) may not migrate into or resolve on the gel. To circumvent this possible limitation, complementary protein profiling analyses of iTRAQ or label free quantitation can be used.
- Recommended sample amount is 50 to 100µg
- Wu, T.L., (2006) Two Dimensional Difference Gel Electrophoresis, in New and Emerging Proteomics Techniques (Nedelkov, D., Nelson, R.W., eds) Vol. 328, Chapter 5 pp 71- 96. Humana Press Inc.,. Totowa, N.J.,