Biology Reference
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from two different samples are mixed, spotted,
and separated on the same gel plate. In 1997,
Ünlü et al. 6 realized the advantages and limita-
tions of 2D-PAGE and developed what is known
as two-dimensional differential in-gel electro-
phoresis (2D-DIGE) to eliminate reproducibility
issues and achieve better protein-to-protein
comparison and quantitation. In a typical exper-
iment, equal amounts of proteins extracted from
two different biological samples, healthy and
diseased, and an internal standard (a pooled
sample formed from mixing equal amounts of
the proteins extracted from the two test samples)
are covalently labeled, each with a cyanine
complex protein mixture fractionation and the
con
rmation of a protein identity by comparing
the migration time with that of a known standard
and comparison of MS spectra of a test protein to
its known protein standard. As a separation,
detection, and quantitation technique, 2D-DIGE
is very useful for measuring protein expression
levels and has played an important role in disease
biomarker discovery. The 2D-PAGE and
2D-DIGE approaches are easily accessible to
most laboratories and possess high resolving
power for the detection of hundreds of proteins
on a single gel plate. Besides detection and quan-
titation, gel electrophoresis can provide informa-
tion about the charge, molecular weight, and
conformational state of a protein. However,
sample-to-sample and day-to-day reproduc-
ibility has been an issue with 2D-PAGE. Resolu-
tion in 2D-PAGE has been greatly improved by
the introduction of immobilized pH gradient
strips (IPGs), which enable the analyst to tailor
the pH gradient for maximum resolution using
ultrazoom gels with a narrow pH gradient range.
With advances in 2D-PAGE, it is not unusual to
resolve two proteins that differ by 0.001 pI units.
The introduction of 2D-DIGE contributed
immensely to solving problems of reproduc-
ibility and quantitation. The use of imagers and
computers allows not only fast data mining,
acquisition, and analysis but also spot detection,
normalization, protein pro
uo-
rescent dye that has a different excitation and
emission wavelength. The same protein from
different samples labeled with any of the dyes
will co-migrate to the same position on the gel
because the dyes were designed to ensure that
proteins common to both samples have the
same relative mobility regardless of the dye
used to tag them. 6 The control sample should
contain every protein present across all samples
in an experiment. This requirement means that
every protein in the experiment has a unique
signal in the internal standard, which is used
for direct quantitative comparisons within each
gel and to normalize quantitative abundance
values for each protein between gels. Scanning
the gel at the speci
c excitation and emission
wavelengths of each dye, using a
uorescence
imager, allows visualization of the differentially
labeled proteins ( Figure 1 ) without further pro-
cessing. The images are merged and analyzed
using software that enables differences between
the abundance levels of proteins to be compared.
ling, background
correction, and data reporting. The advantage
of 2D-DIGE is that the experiment is performed
under the same experimental conditions (pH
gradient and
field strength) using a single gel
plate, which means that inconsistencies between
gels are eliminated, which ensures more accurate
quantitation than if samples are run on separate
gels. 7 Also, 2D-DIGE requires 50% fewer gels,
making it more economical. In addition, less
time is required to detect the protein spots
because the labeling reaction in 2D-DIGE is
faster than visualization using staining methods.
Also, 2D-DIGE is the method of choice when the
absolute protein expression is required. 8
STRENGTHS AND WEAKNESSES OF
2D-PAGE AND 2D-DIGE
Gel electrophoresis is an excellent technique
that has undergone several advances, resulting
in enhanced resolution, detection, quantitation,
and reproducibility. 2D-PAGE can be used for
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