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will experience a broad pH range during isoelectric focusing. This
latter consideration eliminated fl uorescein, the most popular fl uo-
rescent dye of the day. The dyes should not change the charge of
the protein to which they were attached, which raised the issue of
how to attach the dyes to the proteins. The two most reactive
amino acid residues are lysine and cysteine. Lysine's primary
amine provided an easy route to coupling the dye to the protein, but
the coupling process eliminates lysine's positive charge. This meant
that the DIGE dyes had to have an amino group like lysine or an
intrinsic positive charge. Given the p
K
a of the
e
-amino group of
lysine and the 3-10 pH range of isoelectric focusing, a quaternary
amine was a reasonable substitute for a primary amine. Alternatively,
one could couple the DIGE dyes via cysteine residues. At that
point in time, it was not known what fraction of proteins had at
least one cysteine, and I was frankly unfamiliar with the coupling
chemistry, so I put cysteine labeling on the backburner.
Since the literature search did not yield suitable DIGE dyes, I tried
to design and synthesize my own dyes, but my chemistry knowl-
edge was too limited. So I sent a letter to Kodak asking for help
synthesizing the dyes. I got a nice letter back from their lawyers
saying that they would help as long as I signed a form releasing my
invention rights to Kodak. This was before the biotechnology
industry took hold, and academic institutions rarely had technology
transfer offi ces. I was in uncharted territory. This also coincided
with my changing research advisors and a major shift in my research
direction. Consequently, I left the idea behind for 10 years, during
this period I completed my PhD in DNA replication and my post-
doctoral training in cell and developmental biology. In retrospect,
this hiatus was a good thing. In 1981, fl uorescence imaging sys-
tems were too insensitive, and protein sequencing was only done
by Edman sequencing—mass spectrometric protein identifi cation
was only in its infancy.
In 1991, I accepted a faculty position at Carnegie Mellon
University because of its reputation for interdisciplinarity and
entrepreneurship. My lab primarily studies Drosophila embryo
development, particularly how cells change shape during develop-
ment. We were studying a specifi c cell shape change that had been
extensively analyzed by genetic dissection. I was puzzled by the
fact that none of the genetically identifi ed genes required for this
cell shape altering process involved the cytoskeleton or its regula-
tors. I reasoned that cytoskeleton was required for so many cellular
functions that mutations in these genes would prevent the embryo
from developing far enough. But I was certain that changes in the
cytoskeleton and other proteins must occur during this process.
This led me to dust off my old notebooks and reinvestigate DIGE.
An equally important impetus for revisiting DIGE was that my lab
was around the corner from Alan Waggoner's, the father of cyanine
2.4. The Synthesis
of the First DIGE Dyes
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