Biology Reference
In-Depth Information
than previously possible. In 2D-PAGE and
2D-DIGE, the area and intensity of the spots
indicate the levels of protein expression in the
sample. This measure is used to quantitatively
compare protein concentration levels between
two different samples. Also, in-gel digestion of
proteins with trypsin prevents any losses due
to sample manipulation and simpli
to cylindrical tube gels. The
flat slab provided
maximum surface area for cooling the gel and
the resulting patterns were easier to quantify.
Also, a large number of samples can be pro-
cessed using a single 20
20 cm gel plate, allow-
ing the direct comparison of samples processed
under identical conditions. An additional advan-
tage of
es the iden-
flat bed electrophoresis is that it permits
the application of 2D separations.
Current 2D electrophoretic separations of pro-
teomes are based on the method of O
ti
cation of proteins since peptides are easier to
analyze by MS than proteins. This chapter is
a brief introduction to DIGE and its application
to protein biomarker discovery.
Farrell,
5
which was introduced in 1975 for separating
cellular proteins under denaturing conditions.
This method enabled the resolution of hundreds
of proteins on a single gel plate. The principle
employed is very simple: proteins are resolved
in the
'
GEL ELECTROPHORESIS:
H
ISTORICAL PERSPECTIV
E
first dimension according to their isoelec-
tric point and in the second dimension according
to their molecular mass. Today, 2D-PAGE and
its newer version, 2D-DIGE, are the analytical
methods of choice by biologists and biochemists.
To properly understand the advances made in
2D-PAGE, one needs to go back to 1930, when
Arne Tiselius introduced the moving boundary
method as an analytical tool for studying the
electrophoresis of proteins.
1
Since his pioneering
work, for which he received the Nobel Prize,
various forms of electrophoresis have been
employed to separate complex protein mixtures.
Early studies used a single run of gel electropho-
resis that did not result in the complete separa-
tion of complex protein mixtures. Scientists
recognized that a combination of two orthogonal
electrophoretic processes on a gel at right angles
should give a much greater degree of resolution
than is possible with either separately.
2
e
4
Their
prediction was proven true and has formed the
basis for the development of multidimensional
methodologies for the separation of complex
protein mixtures not only by gel electrophoresis
but also by chromatography and capillary elec-
trophoresis. As early as 1962, Raymond and
Aurell
3
demonstrated the signi
TWO-DIMENSIONAL
DIFFERENTIAL IN-GEL
ELECTROPHORESIS
The advantage of 2D-PAGE as a separation
technique is not only the separation of large
numbers of proteins but the determination of
their relative abundances. For example, proteins
extracted from two serum samples (healthy and
diseased) are loaded on a separate gel plate.
After separation and staining, the protein spots
are aligned and scanned to measure their indi-
vidual intensities. Although many advances in
software alignment tools have been made, it
has been challenging to ensure direct spot-to-
spot intensity comparison between two separate
gels because slight differences in gels
cant nonlinear
effects of gel concentration on the electrophoretic
mobility of proteins by employing two-
dimensional (2D) electrophoresis using different
acrylamide gel concentrations to separate serum
proteins. Two years later, Raymond
4
demon-
strated the superiority of
composi-
tion, pH gradient, and applied voltage affect
reproducibility, making it dif
'
cult to compare
protein expression levels between two samples.
To overcome the reproducibility issues, it would
be more accurate if equal amounts of proteins
flat slab gels compared
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