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Fig. 4. Example for difference analysis on the level of a single protein spot. Image data was obtained from fi ve individual
gels (“Pos 1-5”, left image ) and arranged into three groups (“IPS”, “control” and “treated”) within an MFA experiment. The
spot volumes were normalized on the IPS, and the average volumes (“Avg”) of “IPS”, “control” and “treated” were deter-
mined. Referring to the IPS average volume, the abundance variation factors (“Fac”) was calculated. The individual volume
information for all spots included in a MFA experiment can be displayed as peaks in 3D view.
original images. As all spot patterns originate from the consensus
pattern on the fused image, spot matching without matching ambi-
guities can be achieved.
The major challenge of a multifl uorescence analysis is to fi nd
biologically signifi cant changes between different samples. One
big advantage of this technique is the opportunity to link image
data derived from a huge number of gels of a single MFA experi-
ment via the internal standard images. The benefi t of using internal
protein standards (IPS) in complex multifl uorescence 2DE-based
studies has been emphasized in several publications ( 4 ).
For software-based detection and quantifi cation of differences
between two protein samples (e.g. “untreated” vs. “treated”), all
gel images of these samples have to be assigned to two specifi c
groups. A third group contains the images of the IPS. The analysis
software can automatically normalize and quantify the sample spot
volumes in relation to the IPS spot volume. An example for
analysing the spot values obtained from a set of multifl uorescence
2D gels is depicted in Fig. 4 .
To enhance the statistical capabilities of the basic 2D image
analysis software packages, several tools for an extended data analysis
are available. These tools provide additional analysis concepts like
principle component analysis or discriminant analysis as well as
enhanced opportunities for a comprehensive visualization and
presentation of the statistical results.
3.3.3. Difference Analysis
4. Notes
1.
In case of using “traditional” separation gel with 12.5% T and
3% C, gels can be stored in their cassettes for several hours at
RT and overnight at 4°C without losing spot quality.
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