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use of chaotropic agents and the reduction/
alkylation processes all contributed to largely
improved reproducibility of two-dimensional
electrophoresis as well as the overall protein
resolution.
8
The latter aspect
d
low-abundance
protein detection
d
was studied with the goal of
reducing the protein dynamic concentration
range as discussed in the section on low-
abundance proteins.
Among the dif
j
h
f
i
e
c
g
d
b
a
culties of two-dimensional
electrophoresis technology, sensitivity and repro-
ducibility were and still are repeatedly reported.
However, over
1960
1970
1980
1990
2000
Year
FIGURE 1
Main evolutionary breakthrough facts in two-
dimensional electrophoresis with respect to resolution (white
circles) and sensitivity (black circles). The size of the circles
represents the importance of advancements. a: First attempts
with low discrimination between dimensional parameters.
3
b: Improvement using mobility and molecular mass as
discriminating parameters.
4
cations
contributed to an increase in the detection sensi-
tivity by using different staining approaches.
Coomassie Blue was progressively replaced by
colloidal Coomassie with a signi
time,
several modi
cant gain in
sensitivity
9
allowing protein detection at micro-
gram scale and hence a reduction of the sample
need for a two-dimensional plate. Silver staining
was used to detect protein spots
10
with an excel-
lent nanogram and even subnanogram level of
sensitivity. Then dyes with
rst
dimension) coupled orthogonally with molecular mass
(second dimension).
5
d: Coomassie staining. e: Silver stain-
ing.
10
f: Introduction of immobilized pH gradient.
7
g:
Incremental sensitivity of gel staining with colloidal Coo-
massie Blue.
9
h: Improvements in protein solubilization and
spot resolution with detergents.
8
c:
Isoelectric focusing (
i: Improvements in detec-
uorescent dyes.
12
j: 2D-DIGE as a means of
comparing two samples on the same plate.
14
fluorescent properties
advantageously replaced visible dyes with
a good enhancement of spot detectability
11
; these
dyes were also fully compatible with mass spec-
trometry analysis. Fluorescent staining molecules
allowed detecting protein traces as little as 1 to
2 ng with very a good linear dynamic range.
12
Reproducibility of 2-DE is also reported as
a problem to the point that several two-
dimensional gels are operated in order to
average and correct the localization of the spots
manually or with the help of software. To elimi-
nate the reproducibility issue, two-dimensional
differential in-gel electrophoresis (2D-DIGE)
was developed with a concomitant enhancement
of sensitivity, allowing a direct comparison of
two samples on the same gel plate.
13
Proteins
from two different samples are derivatized
each with a different
tion with
presence of SDS. However, distortion of protein
bands, electroendoosmosis, very long migration
times, and diffusion of protein bands during
SDS equilibrium massively contributed to varia-
tions of the spot positioning throughout the
pattern. The introduction of immobilized pH
gradients obtained by copolymerization of
selected ionic acrylamide monomers
6
allowed
a real breakthrough of 2-DE. With the advent of
supported preconstructed pH-gradients,
7
the
technology became increasingly available to bio-
analytical and analytical laboratories.
In spite of all these developments and progres-
sive improvements over time (
Figure 1
), the tech-
nology was still inapplicable to the analysis of
hydrophobic and low-abundance proteins. These
two distinct issues were investigated under
different conditions. The former forced scientists
to
fluorophore, mixed, loaded
on the same gel plate, and separated. Protein
spots are visualized by confocal laser scanning
associated with advanced software capable of
sorting out relative differences in spot intensity.
find better solubilization methods for hydro-
phobic proteins. The addition of detergents, the
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