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comparisons within each cell line A and B (t1A vs. t2A, t1A vs.
t3A, t1A vs. t4A, t2A vs. t3A, t2A vs. t4A, t3A vs. t4A, and
corresponding comparisons in cell line B) plus 16 comparisons
between cell lines can be performed (t1A vs. t1B, t2A vs. t1B,
t3A vs. t1B, t4A vs. t1B, t1A vs. t2B, t2A vs. t2B, t3A vs. t2B,
t4A vs. t2B, t1A vs. t3B, t2A vs. t3B, t3A vs. t3B, t4A vs. t3B,
t1A vs. t4B, t2A vs. t4B, t3A vs. t4B, t4A vs. t4B).
12. For reasons of reproducibility, it should be avoided to have
more than one IPS preparation in the experimental setup. Even
minor deviations in labeling effi ciency may complicate or even
impair a proper evaluation.
13. It should be noted that for reproducible gels, the time needed
for matching is usually low.
14. If thousands of spots are compared using Student's
t
test or
ANOVA for statistical signifi cance, many of these spots may be
statistically different in intensity, from which several may have
achieved this signifi cance by chance alone. The FDR correction
adjusts the Student's
t
test or ANOVA
p
values for each spot to
keep the overall error rate as low as possible.
15. In most cases, the preparative sample should have the same
composition as the IPS. This ensures that each spot which was
identifi ed as differentially abundant in the quantitative analysis
is present in a detectable amount in the preparative gel.
Otherwise, individual spots may be missed, when they are pres-
ent only in a subgroup of samples which was not contained in
the preparative gel. However, if all spots relevant for identifi ca-
tion are increased in a subgroup of samples, the preparative gel
is preferentially prepared using this subgroup.
16. If not enough sample material is available for a preparative gel,
it has been suggested to use sample material most similar to the
samples analyzed. For instance, when analyzing laser microdis-
section samples of tumors, surrounding healthy tissue could be
used for preparing the preparative gel (or vice versa, if tumor
material is abundant and healthy tissue is limiting). In any case,
this must be regarded a less than ideal solution, and we strictly
recommend a verifi cation experiment to ensure that the cor-
rect protein has been identifi ed. Verifi cation could be per-
formed by analyzing the corresponding tissues by Western
blotting when appropriate antibodies are available. Alternatively,
an antibody-independent verifi cation could be performed by
selected reaction monitoring (SRM) mass spectrometry (MS),
which is sensitive, specifi c, and precise and requires low
amounts of protein in the same range used for analytical satu-
ration DIGE gels. A description of the SRM MS method is
available in this topic series (
9
).
17. This step is very critical since the reaction may be incomplete if
the solution is not homogenous. Incomplete reaction will
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