4. Sodium dodecyl sulfate (SDS): 10% solution in double distilled
water. 100 g SDS, add circa 700 mL ddH 2 O, stir the solution
at least 10 m, adjust pH to 6.8 with 1 M HCl, fill up to 1 L with
ddH 2 O, and store at 4
C.
5. Ammonium persulfate (APS): 10% solution in double distilled
water. Store for at most a month.
6. 30% acrylamide/Bis solution: 290 g acrylamide and 10 g
bis-acrylamide; fill up to 1 L with ddH 2 O, filter through a
0.2 μm filter, and store at 4
C.
7. Sample buffer (Laemmli buffer): 250 mM Tris–HCl, pH 6.8,
10% SDS, 50% glycerol, 0.5% bromophenol blue; fill up to
30 mL with ddH 2 O, and store at À20
C. Add 5%
β-mercaptoethanol before use.
8. 5Â running buffer: 1.92 M glycine, 0.25 M Tris Base; add circa
800 mL ddH 2 O and 50 mL 10% SDS, adjust pH to 8.3 with
acetic acid, and fill up to 1 L.
9. Tetramethylethylenediamine (TEMED).
2.6 Immunoblotting
1. A Western blot transfer system and a chemiluminescence imaging system.
2. Transfer buffer: 100 mL of 10Â Transfer buffer (stock 0.25 M
Tris Base, 1.92 M glycine, 1% SDS), 200 mL of ethanol,
700 mL ddH 2 O.
3. PBS.
4. Blocking buffer: PBS, 5–10% non-fat dry milk, 0.05%
Tween 20.
5. Wash buffer: PBS, 0.05% Tween 20.
6. Nitrocellulose membrane.
7. Sponges.
8. Filter paper.
9. Chemiluminescent reagent kit for Western blot.
3 Methods
3.1 Serum
Susceptibility Assay
1. Harvest leptospires by centrifugation at 5500 Â g for 25 min.
Discard supernatant, and wash the pelleted bacteria in 20 mL
of PBS. Collect the cells by centrifugation as described above.
2. Resuspend the sedimented cells in 5 mL of PBS, and count the
number of bacteria by dark-field microscopy using a bacterial
counting chamber as follows:
The number of leptospires per mL ¼ number of bacteria
counted∗ Â dilution (if used) Â 50,000∗∗
192
Lourdes Isaac and Angela Silva Barbosa
water. 100 g SDS, add circa 700 mL ddH 2 O, stir the solution
at least 10 m, adjust pH to 6.8 with 1 M HCl, fill up to 1 L with
ddH 2 O, and store at 4
C.
5. Ammonium persulfate (APS): 10% solution in double distilled
water. Store for at most a month.
6. 30% acrylamide/Bis solution: 290 g acrylamide and 10 g
bis-acrylamide; fill up to 1 L with ddH 2 O, filter through a
0.2 μm filter, and store at 4
C.
7. Sample buffer (Laemmli buffer): 250 mM Tris–HCl, pH 6.8,
10% SDS, 50% glycerol, 0.5% bromophenol blue; fill up to
30 mL with ddH 2 O, and store at À20
C. Add 5%
β-mercaptoethanol before use.
8. 5Â running buffer: 1.92 M glycine, 0.25 M Tris Base; add circa
800 mL ddH 2 O and 50 mL 10% SDS, adjust pH to 8.3 with
acetic acid, and fill up to 1 L.
9. Tetramethylethylenediamine (TEMED).
2.6 Immunoblotting
1. A Western blot transfer system and a chemiluminescence imaging system.
2. Transfer buffer: 100 mL of 10Â Transfer buffer (stock 0.25 M
Tris Base, 1.92 M glycine, 1% SDS), 200 mL of ethanol,
700 mL ddH 2 O.
3. PBS.
4. Blocking buffer: PBS, 5–10% non-fat dry milk, 0.05%
Tween 20.
5. Wash buffer: PBS, 0.05% Tween 20.
6. Nitrocellulose membrane.
7. Sponges.
8. Filter paper.
9. Chemiluminescent reagent kit for Western blot.
3 Methods
3.1 Serum
Susceptibility Assay
1. Harvest leptospires by centrifugation at 5500 Â g for 25 min.
Discard supernatant, and wash the pelleted bacteria in 20 mL
of PBS. Collect the cells by centrifugation as described above.
2. Resuspend the sedimented cells in 5 mL of PBS, and count the
number of bacteria by dark-field microscopy using a bacterial
counting chamber as follows:
The number of leptospires per mL ¼ number of bacteria
counted∗ Â dilution (if used) Â 50,000∗∗
192
Lourdes Isaac and Angela Silva Barbosa