Biology Reference
In-Depth Information
(c) Quantitative DIGE: These applications require an internal
standard, dye swapping, and a suffi cient number of biological
replicates. We have used quantitative 2D DIGE in two more
extensive studies involving rodent animal models. Macrophages
from wild-type and Tyk2-defi cient mice were compared in
their protein patterns before and after treatment with lipopoly-
saccharide (LPS). Five of the differentially regulated proteins
with interesting protein expression patterns in the genotypes
were further investigated at the mRNA as well as the protein
level (immunoblots). Data showed that the lack of a single
protein brought about changes in proteins from different func-
tional categories, suggesting involvement of this tyrosine kinase
in various cellular processes (
8, 15
). In a rat model of endotoxic
shock, the infl uence of LPS on the proteome of different organ-
elles was studied and changes of the protein levels correlated to
functional, genomic, and histological fi ndings. Organelles are
affected to a different degree, but fi rst results suggest major
impairment of endoplasmic reticulum, based also on changed
expression levels of proteins with functions in protein folding,
transport, and detoxifi cation (
6, 16
). Conventional 2DE would
not have been suffi ciently sensitive to detect all the DIGE-
detected candidates and their regulations, in both studies.
4. Notes
1. In practice, 200 mL of separation gel buffer (1.5 M Tris-HCl
pH 8.8) per 10 mL of fi nal solution volume is used. This gives
almost the correct pH, but should be fi ne-adjusted if necessary.
2. DMF can be kept in a desiccator to prevent water uptake and
extend shelf-life.
3. Carrier ampholytes are highly viscous solutions. Take care to
pipette slowly and to use pipette cones, which are not too small;
you may also cut tips to the right size with sharp scissors.
4. Acrylamide monomers are neurotoxic. They should be handled
with care (gloves; use fume cabinet if you prepare solutions
from solid chemicals). Polyacrylamide is only toxic if polymer-
ization is incomplete and the gel still contains monomers.
5. Carrier ampholytes from a different source may be used, either
broad-range (pH 4-10) alone or spiked/supported with narrow-
range ampholytes (e.g., pH 4-6). This largely depends on the
brand used and on the sample.
6. A trace of bromophenol blue may be added to the re-swelling
solution. The migration of this dye toward the anode can then
be checked during the IPG run.
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