Proteomic Analysis of Proteins Secreted by Streptococcus pyogenes - Bacterial Pathogenesis: Methods and Protocols - page 16

H 2 O. Stir until all reagents are dissolved, which may take1horlonger. Do
not heat the solution ( see Note 6 ). Dispense 1-mL aliquots into microcentrifuge
tubes and store at -20 °C. Just prior to use, thaw the SR solution and add 75 m M
dithiothreitol (DTT) ( see Note 7 ).
4. 1× SDS electrophoresis buffer: 25 m M Trizma base, 0.192 M glycine, 0.1% (w/v)
SDS ( see Note 8 ).
5. Agarose sealing solution: 1× SDS electrophoresis buffer, 0.5% (w/v) agarose NA
(GE Healthcare Life Sciences), and a trace of bromophenol blue ( see Note 9 ).
6. SDS equilibration buffer: 50 m M Tris-HCl, 6 M urea, 2% (w/v) SDS, 30%
(v/v) glycerol, and a trace of bromophenol blue. Dispense 10-mL aliquots of the
solution into tubes and store at -20 °C until needed. Just before use, thaw and
add DTT (100 m M final concentration).
7. Broad-range SDS-polyacrylamide gel electrophoresis (SDS-PAGE) molecular
weight standards (Bio-Rad Laboratories).
8. Sample Application Pieces (GE Healthcare Life Sciences).
2.5. Protein Detection, Quantitation, and Analysis
1. SYPRO Ruby protein stain (Bio-Rad Laboratories).
2. Fixer and destain solution: 10% (v/v) methanol and 6% (v/v) glacial acetic acid.
3. 2-D Quant Kit (GE Healthcare Life Sciences).
4. Typhoon 9410 imager (GE Healthcare Life Sciences).
3. Methods
3.1. Isolation of CSPs
1. Culture S. pyogenes in 40 mL of broth media in 50-mL polypropylene centrifuge
tubes at 37 °C for 18 h ( see Note 10 ).
2. Centrifuge at 3220 × g for 10 min at 4 °C to pellet the bacteria.
3. Pass the supernatant fluid through a 0.2-μm syringe filter into a sterile 50-mL
polypropylene centrifuge tube. Proceed to next step or store the supernatant fluid
at -80 °C ( see Note 11 ).
4. Incubate freshly filtered unfrozen supernatants at -20 °C for 30 min ( see Note
12 ). If the supernatant has been frozen, thaw it at room temperature and proceed
to next step when the solution is completely thawed but ice cold.
5. Keep samples on ice during steps 6-41 .
6. Add 100% ice-cold TCA to a final concentration of 10% (v/v) and invert the
tube several times to mix.
7. Add ice-cold acetone to a final concentration of 5% (v/v).
8. Invert tube several times to mix ( see Note 13 ).
9. Incubate the tube at -20 °C for 1 h inverting several times every 20 min.
10. Centrifuge at 6000 × g for 20 min at 4 °C.
11. Discard supernatant fluid in a hazardous waste container.
12. Add 3 mL of ice-cold ethanol to the pellet and resuspend the pellet by vortexing.