DIGE: Past and Future - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 4

2. The Past

DIGE (which my lab pronounces as dee-gay, owing to the Turkish
heritage of Mustafa Ünlü, the graduate student who synthesized
the fi rst pair of DIGE dyes) was initially conceived when I was a
fi rst year graduate student at Albert Einstein College of Medicine.
I was tasked with analyzing a set of Dictyostelium discoideum
temperature-sensitive, motility mutants. In 1981, the Dictyostelium
genome was not known, and there were very few genetics tools for
identifying the affected genes. Two-dimensional gel electrophoresis
(2DE), which was only about 6 years old at the time, seemed like
the best route for discovering the biochemical changes associated
with these mutations. I wanted to compare these cells at the per-
missive and restrictive temperatures. After a couple of abortive tries
at running parallel 2DE gels, I realized that an internal control was
needed. This inspiration probably came from one of the graduate
classes I was taking at the time. The idea was to differentially label
the two samples in separate reactions and then combine the two
reactions so that they could be run on the same 2DE gel. Getting
this idea to work presented several problems: what sort of tags
should be used, how will the tags be attached to the proteins, will
the tags effect how the proteins run on 2DE gels, how would the
tags be detected…?

2.1. The Conception
of DIGE

Proteins could be labeled metabolically in vivo or chemically in vitro.
I wanted to make a versatile, user-friendly method that could be
used on any source of protein. Since only a small number of model
organisms are amenable to metabolic labeling schemes, chemical
labeling after cell lysis seemed to be the most universal approach.
Should the tag be radioactive, colored, or fl uorescent? Radioactivity
was rejected because autoradiography often requires very long
exposures and discrimination between radionuclides can be diffi cult.
Colored tags would only work for very abundant proteins since
color detection requires considerable amounts of absorbing mate-
rial, particular in gels where the path length is 1-2 mm. Fluorescence
seemed like the best choice since one can synthesize compounds
with very different fl uorescent spectra for easy discrimination. Also,
one can detect minute amounts of fl uorescent material, which I
learned from the fl uorescence microscopy we were doing in the
cell biology lab in which I was working.

2.2. What Sort of Tags
Should Be Used?

2.3. The Selection
of Fluorescent Labels

I had no idea what fl uorescent molecules to use for DIGE. I poured
through chemical catalogs and topics on fl uorescent dyes, but none
of the existing fl uorescent compounds really fi t the bill. A key point
in selecting the right pair of dyes was that the dyes should have the
same charge as each other and they should have similar masses.
Another issue was that the dyes should be pH insensitive since they