Proteomic Analysis to Investigate Regulatory Networks in Staphylococcus aureus - Bacterial Pathogenesis: Methods and Protocols - page 30

a defined period (5 min) ( see Fig. 3 ). The resulting autoradiograms reflect the
instantaneous synthesis rates of individual proteins at the time of labeling and can
be identified by using the respective protein master gel. The silver or colloidal
Coomassie-stained protein pattern represents the actual level of proteins accumu-
lated within or outside the cell until the time of harvesting ( see Fig. 3 ).

cultivation

cytolasmic proteins (radioactive)

cytoplasmic proteins extracellular proteins

35 S pulse labeling

Precipitation of
proteins in the
supernatant

Cell disruption
(sonication)

Cell disruption
(ribolvser)

Determination of
protein concentration

Determination of protein concentration

Protein minimal labeling
with CyDyes

2D PAGE

2D PAGE 2D PAGE

Protein detection
Silver staining

Protein detection
Coloidal Coomassie staining

Protein detection
Laser scanning

Protein accumulation
Protein identification

Autoradiogram

Protein accumulation
Protein identification

Protein synthesis/
Protein accumulation

Fig. 3. Strategies of 2D gel experiments for protein identification and for analyzing
protein accumulation and synthesis.