Biology Reference
In-Depth Information
1:1 ratio affecting a large number of spots may indicate an
irreproducible labeling procedure and requires corresponding
improvements.
The optimized labeling protocol established here is used for all
labeling reactions for the saturation DIGE experiment. It should
be applied without any further changes in either the labeling pro-
cedure or the sample preparation steps.
3.5. 2D DIGE
Saturation Labeling
of Analytical Samples
A 2D DIGE gel of mammalian samples typically facilitates the
detection and quantifi cation of around 1,500-2,000 spots with a
suffi cient signal-to-noise ratio of the fl uorescence readout. As for
all multianalyte systems, a statistical evaluation of a suffi cient num-
ber of replicates is mandatory to correct for random deviations of
spot intensities between samples and controls. Reference (
8
) gives
an overview about the number of biological replicates necessary
from the statistical point of view. As a rule of thumb, six biological
replicates (see Note 10) should be prepared for the fi nal experi-
ment. It is recommended to prepare at least one additional sample
to facilitate completion of the experiment even if, for whatever rea-
sons, one sample proves to be inadequate upon analysis. This addi-
tional sample must be contained in the IPS. The DIGE saturation
approach facilitates all conceivable comparisons between all sam-
ples which are contained in the IPS, making the design of the
experiment very straightforward (see Note 11):
1.
Prepare and label all samples to be integrated into the 2D
DIGE saturation experiment according to the optimized label-
ing protocol (see Subheading
3.3
) with Cy5.
2.
Prepare an amount of IPS which is suffi cient for the planned
number of samples (or gels) plus an additional 50%, in case
some of the gels fail (see Note 12). Prepare the IPS by combin-
ing aliquots containing the same amounts of protein from each
sample.
3.
Label the IPS with Cy3 according to the optimized labeling
protocol.
4.
Combine each sample with an aliquot of IPS and subject the
mixture to 2D electrophoresis.
5.
Immediately after electrophoresis, scan the gel to generate Cy5
and Cy3 readouts.
3.6. Image Analysis
and Spot
Quantifi cation
In order to detect spots differing in intensity between several gels
of different samples, dedicated software tools are available on the
market. The software packages usually perform (1) the detection
and quantifi cation of all spots in all gels, (2) the inter-gel matching
of these spots, and fi nally (3) the calculation of spot intensity ratios
between gels from different sample groups. For saturation DIGE
experiments, it is recommended to use software packages supporting
the concept of co-migration of proteins labeled with different dyes
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