Proteomic Analysis to Investigate Regulatory Networks in Staphylococcus aureus - Bacterial Pathogenesis: Methods and Protocols - page 33

2. Add 5 mL of freshly prepared 100% (w/v) TCA (end concentration 10% (v/v))
to the supernatant and precipitate the proteins overnight at 4 °C.
3. Centrifuge precipitate for 1 h and decant the supernatant very carefully. Wash the
precipitate with 20 mL of 100% (v/v) ice-cold ethanol, centrifuge at 8000 × g for
10 min at 4 °C, and decant supernatant. Repeat the washing procedure twice.
4. Afterward, remove the pellet from the tube, mix with 10 mL of 100% ice-cold
ethanol, transferred to a 15-mL Falcon tube, and centrifuge at 8000 × g for
10 min at 4 °C. The supernatant should be decanted very carefully. Repeat this
washing procedure at least six times. One of these steps should be done with
70% (v/v) ice-cold ethanol instead of 100% ice-cold ethanol. After decanting the
supernatant, dry the protein pellet at room temperature.
5. Dissolve the dried protein pellet in 8 M urea/2 M thiourea and mix by shaking
for 20 min at room temperature. Centrifuge the solution for 15 min at 21,000 × g
and transfer the supernatant into a new microtube.
6. Determine protein concentration by using ROTI-Nanoquant (Roth) ( see Note 3 ).
Store protein solution at -20 °C.
3.2.3. Preparation of Cytoplasmic Protein Extracts Labeled
With [ 35 S]- L -Methionine (Pulse-Labeling Reaction)
1. Grow cells in synthetic medium at 37 °C to an OD of 0.5.
2. Transfer 10 mL of the culture volume to a new Erlenmeyer flask and add 10 μL
of (100 μCi) l-[ 35 S]-methionine to the culture. Stop the labeling reaction after
5 min by adding 1 mL of stop solution and by transferring the Erlenmeyer flask
to ice.
3. Pellet cells by centrifugation at 8000 × g for 5 min at 4 °C.
4. Wash cell pellets twice with 1 mL of ice-cold TE buffer and resuspend in 400 μL
of TE buffer.
5. For cell lysis, add 10 μL of lysostaphin solution (10 mg/mL) to the cell suspension.
6. After incubation on ice for 10 min, disrupt cells by sonication. For this step,
place a beaker of ice water around the sample tube to keep it cold. Sonicate the
samples for 1 min (0.5/s, low) followed by a 1-min cooling break. Repeat this
process three times. Sonication is complete when the solution appears noticeably
less cloudy than the starting solution.
7. After sonication, centrifuge the sample at 21,000 × g for 10 min at 4 °C. Transfer
the supernatant to a new tube and repeat the centrifugation process for 30 min.
8. Determine protein concentration by using ROTI-Nanoquant (Roth) ( see Note 3 ).
Store the protein solution at -20 °C.
3.3. CyDye DIGE Minimal Labeling (for Extracellular Proteins)
1. The recommended concentration of protein is between 5 and 10 mg/mL.
2. The pH of the protein solution should be between pH 8.0 and 9.0. Check by
spotting 1 μL on a pH indicator strip. If the pH is lower than 8.0, then the pH