Proteomic Analysis to Investigate Regulatory Networks in Staphylococcus aureus - Bacterial Pathogenesis: Methods and Protocols - page 39

induction induction

Unchanged
synthesis

Unchanged
synthesis

repression repression

20' after shift to
anaerobic conditions

accumulation
(silver staining)

synthesis
( 35 S labeling)

Fig. 5. Dual-channel imaging technique. Bacterial cells were grown in synthetic
medium without nitrate to an optical density at 500 nm (OD 500 ) of 0.5. The proteins
were labeled with [ 35 S]-l-methionine 20 min after transfer to anaerobic conditions.
Protein extracts were separated on 2D gels. The resulting images from the protein
synthesis ([ 35 S]-l-methionine labeling) and the protein accumulation (staining with
silver nitrate) were overlaid in “dual channel images.” Proteins whose synthesis was
induced in response to the stressor but have not accumulated yet are colored red,
repressed spots that are still present but not synthesized anymore are colored green, and
spots without changed expression behavior in response to stress are shown in yellow.
( See Color Plate 3, following p. 46.)

starvation stimulus are colored red. Looking for such red-colored proteins is
a simple approach for visualizing all proteins induced by a single stimulus,
thereby defining the entire stimulon. Proteins repressed by the stimulus can also
be visualized by this powerful technique. Green-colored proteins that are no
longer synthesized (no longer red) but still present in the cell are the candidates
for repression by the stimulus. For a more detailed study, the synthesis rate of
the proteins after exposure to stress can be quantified ( see Fig. 5 ).
By using the Ettan DIGE, a pooled internal protein standard labeled with Cy2
can be incorporated on every 2D gel. Normally, the internal standard represents
a mix of all protein extracts of an experiment and is used to match the protein