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Quantitative Proteomics Center
Such a gel can then be subjected to
image analysis and protein spots are excised from the gel and
processed. These proteins can be identified by a combination of mass
spectrometry and database searching, and in many cases hundreds of
proteins of interest are identified:
In difference gel electrophoresis (DIGE),
two or more protein samples are labeled with different cyanine dyes and
then separated by 2D gel electrophoresis. A laser fluorescence image is
recorded which is composed of two sub-images; these overlap completely
on a pixel-to-pixel basis. Differentially expressed proteins are easily
detected in such an image, as in this example where the two
sub-images are alternately flashed on the screen and the differentially
expressed proteins appear in only one or the other sub-image:
In an enhanced version of this
technique , the protein samples to be compared are labeled with
different cyanine dyes and subsamples are also mixed with an internal
standard in which all protein extracts in the experiment are pooled and
labeled with a third dye. The overall workflow for this technique is illustrated in this diagram:
This technology has been applied to
basic, applied and clinical research supporting studies of cell and
microbial cultures, animal, plant and human tissues (Lilley and
Friedman, 2004; Marouga et al., 2005). Interest in this technique has
steadily increased since it was first described by Minden’s group at
Carnegie-Mellon (Unlu and Minden, 1995; Unlu et al., 1997). Also see
examples of the application of this technique in our publications.
References
Lilley KS, Friedman DB (2004) All about DIGE: quantification technology for differential-display 2D-gel proteomics. Expert Review of Proteomics 1:401-409.
Marouga R, David S, Hawkins E (2005) The development of the DIGE system: 2D fluorescence difference gel analysis technology. Analytical and Bioanalytical Chemistry 382:669-678.
Unlu M, Minden J (1995) DIFFERENCE GEL-ELECTROPHORESIS. Molecular Biology of the Cell 6:2596-2596.
Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis 18:2071-2077.
References
Lilley KS, Friedman DB (2004) All about DIGE: quantification technology for differential-display 2D-gel proteomics. Expert Review of Proteomics 1:401-409.
Marouga R, David S, Hawkins E (2005) The development of the DIGE system: 2D fluorescence difference gel analysis technology. Analytical and Bioanalytical Chemistry 382:669-678.
Unlu M, Minden J (1995) DIFFERENCE GEL-ELECTROPHORESIS. Molecular Biology of the Cell 6:2596-2596.
Unlu M, Morgan ME, Minden JS (1997) Difference gel electrophoresis: A single gel method for detecting changes in protein extracts. Electrophoresis 18:2071-2077.
Here are some examples of studies that we have done with this technique:
