Proteomic Analysis to Investigate Regulatory Networks in Staphylococcus aureus - Bacterial Pathogenesis: Methods and Protocols - page 32

3.1. Bacterial Growth Conditions
1. For radioactive pulse-labeling experiments, cultivate bacterial strains in 50 mL of
synthetic medium without methionine (11) . For 100 mL synthetic medium, add
4 mL of 25× MOPS buffer, 14 μL of 1 M citrate, 740 μL of 20% glucose, 2 mL
of each amino acid mixture, 100 μL of each vitamin solution, 100 μL of each
trace element solution, 150 μL of FeCl 2 , and 150 μL of 2 N NaOH to 50 mL of
2× basic medium. Afterward, bring the volume of the medium to 100 mL with
sterilized, deionized water. Inoculate the synthetic medium with exponentially
growing cells of the appropriate S. aureus strain (grown overnight to start a fresh
culture) in the same medium to an initial optical density at 500 nm (OD 500 )of
0.07-0.1. Cultivate cells with vigorous agitation at 37 °C.
2. For preparation of unlabeled cytoplasmic and extracellular protein extracts,
inoculate 100 to 1500 mL of complex medium (TSB or LB) or synthetic medium
with exponentially growing cells of the appropriate S. aureus strain to an initial
OD 540 of 0.05. Grow cells with vigorous agitation at 37 °C.
3. For stress kinetic experiments, inoculate 100 mL of medium with exponentially
growing cells of the appropriate S. aureus strain to an initial OD 540 of 0.05. At an
OD of 0.5-0.7, transfer 10-50 mL of cell culture to new, preheated Erlenmeyer
flasks and expose to stress conditions. Harvest cells at fixed time intervals after
imposition of stress. As a control, harvest 20-50 mL from the untreated culture
immediately before and at the end of the stress experiment.
3.2. Protein Preparation
3.2.1. Cytoplasmic Proteins
1. For preparationof cell extracts at differentODs (OD 540 = 0.5 and10) or different time
points after impositionof stress, separatecellsof 50-mLcultures fromthe supernatant
by centrifugation at 7000 × g for 10 min at 4 °C. (Simultaneously, the supernatant
can be used for preparation of extracellular proteins [ see Subheading 3.2.2. ].)
2. Wash cell pellets with 1 mL of ice-cold TE buffer, resuspend in 1 mL TE buffer,
and transfer into screw top tubes containing 500 μL of glass beads.
3. Disrupt cells by homogenization with glass beads using the Rybolyser (Thermo
Electron Corporation) for 30 s at 6.5 m/s.
4. Centrifuge lysate at 21,000 × g for 25 min at 4 °C to remove cell debris and then
transfer supernatant to a new tube.
5. Centrifuge at 21,000 × g for 45 min (all at 4 °C) to remove insoluble and aggre-
gated proteins, which disturb the isoelectric focusing (IEF) of the proteins.
6. Determine protein concentration by using Roti-Nanoquant (Roth) ( see Note 3 ).
Store the protein solution at -20 °C.
3.2.2. Extracellular Proteins
1. Separate the supernatant of a 50-mL culture from cells by centrifugation (10 min,
7000 × g , 4 °C) and transfer into a new tube. (Optional: centrifugation step can
be repeated if there are cells remaining in the supernatant.)