bacterial membrane components. Both protocols will be detailed
hereafter, and analysis methods will also be reviewed.
2 Materials
2.1 Specific
Reagents for LPS
Extraction and
Analysis
1. Bacterial culturing medium Ellinghausen-McCullough-Johnson-Harris (EMJH): 1.0 g/L disodium phosphate, 0.3 g/L
monopotassium phosphate, 1.0 g/L sodium chloride, 0.25 g/
L ammonium chloride, 5 mg/L thiamine. EMJH medium is
supplemented with 10 g/L serum bovine albumin, 4 mg/L
heptahydrate zinc sulfate, 50 mg/L heptahydrate iron sulfate,
15 mg/L hexahydrate magnesium chloride, 15 mg/L dihydrate sodium chloride, 0.1 g/L sodium pyruvate, 0.12 mg/L
B12 vitamin, 0.4 g/L glycerol, 1.25 g/L Tween 80.
2. Polypropylene centrifuge bottles of 250 mL.
3. Dulbecco’s phosphate buffered saline (DPBS): 8 g/L sodium
chloride, 200 mg/L potassium chloride, 1.44 g/L sodium
phosphate dibasic, 240 mg/L monopotassium phosphate,
pH 7.4.
4. Sodium dodecyl sulfate (SDS): 10% or more solution in H 2 O.
5. Ammonium persulfate (APS): 10% solution in H 2 O.
6. Precasted or hand-casted linear gradient gels for
SDS-polyacrylamide gel electrophoresis (SDS-PAGE): 4%
(light solution) to 15% (heavy solution) acrylamide/bisacrylamide (29.2:0.8), 0.375 M Tris–HCl pH 8.8, 5 mL/L of 10%
APS, 0.5 mL/L N,N,N,N
0 -tetramethylethylenediamine.
7. Laemmli
buffer:
4%
SDS,
20%
glycerol,
10%
2-mercaptoethanol, 0.004% bromophenol blue, 0.125 M
Tris–HCl, pH 6.8.
8. SDS-PAGE running buffer: 25 mM Tris–HCl, 192 mM glycine, 0.1% SDS, pH 8.3.
9. Phenol (solid).
10. Endotoxin-free water.
11. Periodic acid (solid).
12. Silver nitrate: 20% solution in H 2 O.
13. Citric acid (solid).
14. Proteinase K.
2.2 Specific
Equipment for LPS
Extraction and
Analysis
1. Incubator with no agitation at 30
C.
2. Freezer À80
C.
3. Water bath at 68
C located under a chemical hood.
4. Neoprene gloves.
Purification of LPS from Leptospira
55
hereafter, and analysis methods will also be reviewed.
2 Materials
2.1 Specific
Reagents for LPS
Extraction and
Analysis
1. Bacterial culturing medium Ellinghausen-McCullough-Johnson-Harris (EMJH): 1.0 g/L disodium phosphate, 0.3 g/L
monopotassium phosphate, 1.0 g/L sodium chloride, 0.25 g/
L ammonium chloride, 5 mg/L thiamine. EMJH medium is
supplemented with 10 g/L serum bovine albumin, 4 mg/L
heptahydrate zinc sulfate, 50 mg/L heptahydrate iron sulfate,
15 mg/L hexahydrate magnesium chloride, 15 mg/L dihydrate sodium chloride, 0.1 g/L sodium pyruvate, 0.12 mg/L
B12 vitamin, 0.4 g/L glycerol, 1.25 g/L Tween 80.
2. Polypropylene centrifuge bottles of 250 mL.
3. Dulbecco’s phosphate buffered saline (DPBS): 8 g/L sodium
chloride, 200 mg/L potassium chloride, 1.44 g/L sodium
phosphate dibasic, 240 mg/L monopotassium phosphate,
pH 7.4.
4. Sodium dodecyl sulfate (SDS): 10% or more solution in H 2 O.
5. Ammonium persulfate (APS): 10% solution in H 2 O.
6. Precasted or hand-casted linear gradient gels for
SDS-polyacrylamide gel electrophoresis (SDS-PAGE): 4%
(light solution) to 15% (heavy solution) acrylamide/bisacrylamide (29.2:0.8), 0.375 M Tris–HCl pH 8.8, 5 mL/L of 10%
APS, 0.5 mL/L N,N,N,N
0 -tetramethylethylenediamine.
7. Laemmli
buffer:
4%
SDS,
20%
glycerol,
10%
2-mercaptoethanol, 0.004% bromophenol blue, 0.125 M
Tris–HCl, pH 6.8.
8. SDS-PAGE running buffer: 25 mM Tris–HCl, 192 mM glycine, 0.1% SDS, pH 8.3.
9. Phenol (solid).
10. Endotoxin-free water.
11. Periodic acid (solid).
12. Silver nitrate: 20% solution in H 2 O.
13. Citric acid (solid).
14. Proteinase K.
2.2 Specific
Equipment for LPS
Extraction and
Analysis
1. Incubator with no agitation at 30
C.
2. Freezer À80
C.
3. Water bath at 68
C located under a chemical hood.
4. Neoprene gloves.
Purification of LPS from Leptospira
55