Proteomic Analysis of Proteins Secreted by Streptococcus pyogenes - Bacterial Pathogenesis: Methods and Protocols - page 15

2. Materials
2.1. Isolation of CSPs
1. 100% trichloroacetic acid (TCA) ( see Note 1 ).
2. Acetone ( see Note 1 ).
3. Absolute ethanol.
4. 25-mm, 0.2-μm syringe filter.
5. 10-mL luer-lock syringe.
6. Broth medium (Todd-Hewitt or chemically defined media [ see Note 2 ]).
7. 10× DNase buffer: 100 m M Tris-HCl (pH 7.5), 25 m M MgCl 2 ,5m M CaCl 2 .
8. RNase A.
9. DNase.
2.2. Isoelectric Focusing
1. IPGphor (GE Healthcare Life Sciences, Piscataway, NJ, USA).
2. 7-cm Immobiline DryStrips (pH 4-7) (GE Healthcare Life Sciences).
3. 7-cm IPG ceramic strip holder (GE Healthcare Life Sciences).
4. Dry-strip cover fluid.
5. IPG buffer (pH 4-7) (GE Healthcare Life Sciences).
2.3. Casting Polyacrylamide Gels
1. 1.5 M Tris-HCl (pH 8.8).
2. 10% (w/v) ammonium persulfate ( see Note 3 ).
3. 10% (w/v) sodium dodecyl sulfate (SDS).
4. Acrylamide solution: acrylamide:bis-acrylamide (37.5:1). Acrylamide is a potent
neurotoxin. Wear gloves and protective clothing when using solutions containing
acrylamide ( see Note 4 ).
5. Mini-PROTEAN 3 large glass plates with 1.0-mm spacers (Bio-Rad Laboratories,
Hercules, CA, USA).
6. Mini-PROTEAN 3 Multi-Casting Chamber (Bio-Rad Laboratories).
7. Mini-PROTEAN 3 short glass plates (Bio-Rad Laboratories).
8. N,N,N´,N´-tetramethylethylenediamine (TEMED).
9. Water-saturated butanol: Mix 50 mL of n -, i -, or t -butanol and 10 mL of deionized
H 2 O. Combine in a bottle and shake. Use the top phase to overlay gels. Store at
room temperature.
2.4. Second-Dimension Gel Electrophoresis
1. Mini-PROTEAN 3 Dodeca Cell (Bio-Rad Laboratories).
2. MultiTemp III Thermostatic Circulator (GE Healthcare Life Sciences) ( see
Note 5 ).
3. Solubilization and rehydration solution (SR solution): 8 M urea, 2 M thiourea
(8) , and 4% (w/v) CHAPS. Add dry reagents and fill to volume with distilled