Biology Reference
In-Depth Information
The saturation labeling concept is especially useful when
sample amounts are very limited, i.e., only a few micrograms of
total protein are available per individual sample. If sample amount
is not limiting, the DIGE minimal labeling approach offers clear
advantages with respect to gel number (only half the number of
gels is necessary) and robustness of the labeling reaction.
As a consequence from the concept of labeling sulfhydryl
groups to completion, saturation DIGE requires a strict stoichi-
ometry between reactive sulfhydryl groups present in the lysate
and CyDyes.
The labeling reaction must be carefully optimized for each
kind of sample, since the sulfhydryl content may vary substantially
between proteins from different biological sources, notably also
within different cells, tissues, or body fl uids from one distinct
organism. In addition, the sample preparation procedure used may
infl uence labeling effi ciency since different procedures may lead to
different amounts of substances interfering with the labeling
reaction.
For successful proteomic analyses based on 2D DIGE saturation
labeling, the following preconditions must be fulfi lled: (1) availability
of a reliable and robust protocol to lyse the biological material in a
very reproducible manner, a challenging task especially with minute
sample amounts; (2) availability of a reproducible and robust
protocol for protein quantifi cation from scarce samples; (3) the
ability to conduct 2D PAGE in an excellent manner with respect
to reproducibility and separation power in both dimensions;
and(4) in cases where protein identifi cation is obligatory, the
possibility to generate enough sample(s) for at least one prepara-
tive gel containing a total protein amount in the order of 100-500
g,
depending on sample complexity and intensity of relevant spots, for
mass spectrometry-based protein identifi cation.
This chapter describes:
μ
1. Special considerations for sample preparation in saturation
DIGE.
2. Optimization of the saturation dye labeling reaction.
3. Design of a 2D DIGE study.
4. Evaluation of a 2D DIGE experiment.
5. Identifi cation of protein spots from a preparative saturation
DIGE gel.
It is assumed that the reader is familiar with 2D PAGE using
immobilized pH gradients in the fi rst and SDS PAGE in the
second dimension. Therefore, no descriptions about 2D PAGE in
general or 2D PAGE technical equipment are given.
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