Biology Reference
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Five biological replicates of each mouse genotype/diet were
chosen to obtain some statistical validity. Frozen liver samples
from these animals were solubilised and labelled with one of two
fl uorescent dyes (red, Cy3, and blue, Cy5). Equal amounts of
each of the underivatised samples were pooled and labelled with a
third fl uorescent dye (yellow, Cy2). The samples were then mixed
and subjected to two-dimensional gel electrophoresis (2DE) so
that per gel there were two samples of different fl uorescent labels,
plus an aliquot of the labelled pooled internal standard. Subsequent
fl uorescence scanning of the gels at each of the dyes' excitation/
emission wavelengths yields gel images that can be superimposed, and
in this image spots of red and blue indicate the more predominant
proteins (see Fig. 1 ).
Use of the pooled internal standard across all of the gels allows
the gel imaging software to normalise the response for each gel.
Thus, comparisons can be made to identify the proteins that are
reproducibly either up- or down-regulated between samples. Protein
spots of interest from one of the DIGE gels or a preparative gel,
which is run simultaneously without fl uorescent dye but with an
increased amount of protein, can then be picked, subjected to
tryptic digestion and analysed by LC-MS/MS. Protein identifi ca-
tion is then made by matching actual peptide and fragment masses
with theoretical equivalents from a sequence database.
Fig. 1. Fluorescent DIGE image highlighting the differential proteins expressed for the Swiss Jim Lambert genotype fed on
either a standard (Cy3-labelled, pink ) or a high-fat (Cy5-labelled, blue ) diet (gel 4 of Table 1 ) .
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