2D DIGE Saturation Labeling for Minute Sample Amounts - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 104

p values <0.05 if FDR correction was performed. Moreover, it is
recommended to fi lter for spots which are present in at least three-
quarter of the gels:

1. Filter settings can be set in the BVA module within “Protein
view” (Menu item “Process” → “Protein Filter”).
2. Make sure the “Assign Protein of Interest” box is checked.
3. To display only the spots assigned as “Protein of interest,” set
the view properties to “Protein of interest” (Menu item
“View” → “Properties” → Tab “Protein Table” → “Protein
Table Filter” → “Protein of Interest”).

The Extended Data Analysis (EDA) module of the DeCyder soft-
ware is a tool for multivariate analysis of protein expression data
generated with the BVA module. In addition to the univariate
analyses (Student's t test, One-Way ANOVA and Two-Way
ANOVA) performed with the BVA module, the following analyses
can be performed:

3.6.7. Extended Data
Analysis

Principal component analysis (PCA).

●

Hierarchical clustering.

●

K -means clustering.
Self-organizing maps.

●

●

Gene shaving.

●

Discriminant analysis.

●

To describe all the statistical methods provided by the EDA
module would clearly go beyond the scope of this chapter.
Nevertheless, we want to point out that, especially, the PCA analy-
sis has shown to be a powerful tool to fi nd outlier gels in large gel
sets. Furthermore, hierarchical clustering has demonstrated in
many experiments to be a powerful tool to estimate the grade of
difference between experimental groups and the grade of similarity
of samples within an experimental group.

For mass spectrometry-based identifi cation of relevant proteins,
the amount of protein present on an analytical saturation DIGE
gel (5

3.7. Preparative 2D
DIGE Saturation
Labeling for Protein
Identifi cation

g total protein) is usually not suffi cient. Therefore, a pre-
parative gel, typically containing 100-500

μ

g of total protein, has
to be prepared. To reproduce exactly the spot pattern of the ana-
lytical gel, it is essential to label the protein solution with saturation
dye Cy3 (see Note 15). The stoichiometry of protein and TCEP/
CyDye determined in the optimization experiment has to be main-
tained in the preparative labeling procedure. Since the saturation
labeling procedure for a preparative gel is designed to give a fi nal
volume of 450

μ

L, cup loading of the sample for isoelectric focus-
ing is not possible. Instead, in-gel rehydration sample loading must

μ