5. 50 mL plastic tubes resistant to hot phenol.
6. Dialysis tubes of molecular weight cutoff: 12,000–14,000 Da.
7. Centrifuge: spinning up to 3345 Â g.
8. Ultracentrifuge: spinning up to 100,000 Â g.
9. 2 mL endotoxin-free glass tubes for lyophilization.
10. Lyophilization equipment: freeze dryer with temperature
between À80 and À90
C and pressure between 30 and
100 mTorr.
11. Precision balance.
2.3 Necessary
Buffers for the Silver
Staining Analysis
All the necessary buffers for the silver staining analysis are described
in Table 1.
3 Methods
The purification protocol of the leptospiral LPS involves several
toxic chemicals and takes a lot of time. It is recommended to read
carefully Notes 1–4 before starting the preparation.
3.1 Leptospira
Cultures and
Centrifugation
Leptospira strains are grown in liquid EMJH medium at 30
C and
with no agitation. Small volumes (1–2 mL) can be enough for the
proteinase K protocol, but large volumes are used for the hot
phenol procedure. For the latter, around 2–3 L of late exponential
bacterial cultures are required to get enough LPS to be weighed.
Such cultures can be obtained in the following manner:
Table 1
Composition and indicative volumes for preparation of silver stainning solutions
Name
Composition
Indicative volumes for one mini-gel
Fixation solution
Ethanol 40%
Acetic acid 5%
(1) Endotoxin-free water
27.5 mL
(2) Ethanol (>99%)
20 mL
(3) Acetic acid (>99%)
2.5 mL
Oxydation solution Ethanol 40%
Acetic acid 5%
Periodic acid 3.5 mg/mL
(1) Endotoxin-free water
27.5 mL
(2) Ethanol (>99%)
20 mL
(3) Acetic acid (>99%)
2.5 mL
(4) Periodic acid (solid)
175 mg
Staining solution
NaOH 0.2 N
Ammonim hydroxide 1.4%
Silver nitrate 0.7%
(1) NaOH 1 N
7 mL
(2) Ammonium hydroxide (>99%)
500 μL
(3) Silver nitrate (20%)
1.25 mL
(4) Endotoxin-free water
26.25 mL
Revelation solution Citric acid 0.2 mg/mL
Formaldehyde 0.2%
(1) Endotoxin-free water
49.9 mL
(2) Citric acid (solid)
10 mg
(3) Formaldehyde
100 μL
56
Delphine Bonhomme and Catherine Werts
6. Dialysis tubes of molecular weight cutoff: 12,000–14,000 Da.
7. Centrifuge: spinning up to 3345 Â g.
8. Ultracentrifuge: spinning up to 100,000 Â g.
9. 2 mL endotoxin-free glass tubes for lyophilization.
10. Lyophilization equipment: freeze dryer with temperature
between À80 and À90
C and pressure between 30 and
100 mTorr.
11. Precision balance.
2.3 Necessary
Buffers for the Silver
Staining Analysis
All the necessary buffers for the silver staining analysis are described
in Table 1.
3 Methods
The purification protocol of the leptospiral LPS involves several
toxic chemicals and takes a lot of time. It is recommended to read
carefully Notes 1–4 before starting the preparation.
3.1 Leptospira
Cultures and
Centrifugation
Leptospira strains are grown in liquid EMJH medium at 30
C and
with no agitation. Small volumes (1–2 mL) can be enough for the
proteinase K protocol, but large volumes are used for the hot
phenol procedure. For the latter, around 2–3 L of late exponential
bacterial cultures are required to get enough LPS to be weighed.
Such cultures can be obtained in the following manner:
Table 1
Composition and indicative volumes for preparation of silver stainning solutions
Name
Composition
Indicative volumes for one mini-gel
Fixation solution
Ethanol 40%
Acetic acid 5%
(1) Endotoxin-free water
27.5 mL
(2) Ethanol (>99%)
20 mL
(3) Acetic acid (>99%)
2.5 mL
Oxydation solution Ethanol 40%
Acetic acid 5%
Periodic acid 3.5 mg/mL
(1) Endotoxin-free water
27.5 mL
(2) Ethanol (>99%)
20 mL
(3) Acetic acid (>99%)
2.5 mL
(4) Periodic acid (solid)
175 mg
Staining solution
NaOH 0.2 N
Ammonim hydroxide 1.4%
Silver nitrate 0.7%
(1) NaOH 1 N
7 mL
(2) Ammonium hydroxide (>99%)
500 μL
(3) Silver nitrate (20%)
1.25 mL
(4) Endotoxin-free water
26.25 mL
Revelation solution Citric acid 0.2 mg/mL
Formaldehyde 0.2%
(1) Endotoxin-free water
49.9 mL
(2) Citric acid (solid)
10 mg
(3) Formaldehyde
100 μL
56
Delphine Bonhomme and Catherine Werts