6. Add glacial acetic acid (some drops) to obtain a neutralize
solution (check with a pH paper).
7. Concentrate under air stream.
8. Add 1 volume of CHCl 3 and 1 volume of water.
9. Gently vortex.
10. Transfer the lower organic phase in a pre-weighed glass tube.
11. Dry the lower organic phase containing alkali-resistant lipids
under air or nitrogen stream.
3.9 Enrichment of
Lipid Extract in LongChain Compounds
(TDM, TMM, GroMM, or
MAME) by Methanol
Precipitation
Due to the chain length (60–100 carbon atoms), mycolic acids
(MA) are insoluble in methanol (see Note 4).
1. Dissolve up to 50 mg of total “free” lipids (Subheading 3.1,
item 17) or total fatty acids methyl esters (Subheading 3.3,
item 10) in a minimal amount of chloroform (0.5 mL or a few
drops) in a conic glass centrifuge tube.
2. Add 5 mL of cold methanol until a white precipitate appears.
3. Sit overnight at 4
C.
4. Centrifuge at 4000 Â g for 30 min at 4
C.
5. Transfer the methanolic supernatant with a Pasteur pipette into
a weighed test tube. The pellet contains mainly MA and derivatives (trehalose dimycolate, TDM; trehalose monomycolate,
TMM; and glycerol monomycolate, GroMM).
3.10 Enrichment of
Lipid Extract in
Phospholipids and
Sulfolipids by Acetone
Precipitation
1. Dissolve the total “free” lipid extract (Subheading 3.1, step
17), around 50–100 mg, in a minimum volume of chloroform
in a centrifuge glass tube.
2. Add 5 mL acetone carefully until precipitate appears.
3. Vortex.
4. Sit overnight at 4
C.
5. Centrifuge at 4000 Â g, for 30 min at 4
C.
6. Transfer the methanolic supernatant with a Pasteur pipette to
separate soluble-acetone lipids from the pellet containing insoluble phospholipids in cold acetone. The soluble fraction contains mainly the SGL family [29].
3.11 Purification of
Free Lipids by
Adsorption
Chromatography on
Florisil Column
1. Prepare the column as a slurry of Florisil in the most apolar
solvent, depending on the nature of lipids, e.g., petroleum
ether (apolar lipids) or chloroform (polar lipids). Use an
amount of Florisil equal to around 50-fold the weight of lipids.
2. Place a small piece of cotton at the bottom of a chromatography column to retain the adsorbent.
3. Observe a good ratio between height and diameter (e.g.,
2 Â 40 cm) to ensure a good separation of lipids.
Lipid and Lipoarabinomannan
129
solution (check with a pH paper).
7. Concentrate under air stream.
8. Add 1 volume of CHCl 3 and 1 volume of water.
9. Gently vortex.
10. Transfer the lower organic phase in a pre-weighed glass tube.
11. Dry the lower organic phase containing alkali-resistant lipids
under air or nitrogen stream.
3.9 Enrichment of
Lipid Extract in LongChain Compounds
(TDM, TMM, GroMM, or
MAME) by Methanol
Precipitation
Due to the chain length (60–100 carbon atoms), mycolic acids
(MA) are insoluble in methanol (see Note 4).
1. Dissolve up to 50 mg of total “free” lipids (Subheading 3.1,
item 17) or total fatty acids methyl esters (Subheading 3.3,
item 10) in a minimal amount of chloroform (0.5 mL or a few
drops) in a conic glass centrifuge tube.
2. Add 5 mL of cold methanol until a white precipitate appears.
3. Sit overnight at 4
C.
4. Centrifuge at 4000 Â g for 30 min at 4
C.
5. Transfer the methanolic supernatant with a Pasteur pipette into
a weighed test tube. The pellet contains mainly MA and derivatives (trehalose dimycolate, TDM; trehalose monomycolate,
TMM; and glycerol monomycolate, GroMM).
3.10 Enrichment of
Lipid Extract in
Phospholipids and
Sulfolipids by Acetone
Precipitation
1. Dissolve the total “free” lipid extract (Subheading 3.1, step
17), around 50–100 mg, in a minimum volume of chloroform
in a centrifuge glass tube.
2. Add 5 mL acetone carefully until precipitate appears.
3. Vortex.
4. Sit overnight at 4
C.
5. Centrifuge at 4000 Â g, for 30 min at 4
C.
6. Transfer the methanolic supernatant with a Pasteur pipette to
separate soluble-acetone lipids from the pellet containing insoluble phospholipids in cold acetone. The soluble fraction contains mainly the SGL family [29].
3.11 Purification of
Free Lipids by
Adsorption
Chromatography on
Florisil Column
1. Prepare the column as a slurry of Florisil in the most apolar
solvent, depending on the nature of lipids, e.g., petroleum
ether (apolar lipids) or chloroform (polar lipids). Use an
amount of Florisil equal to around 50-fold the weight of lipids.
2. Place a small piece of cotton at the bottom of a chromatography column to retain the adsorbent.
3. Observe a good ratio between height and diameter (e.g.,
2 Â 40 cm) to ensure a good separation of lipids.
Lipid and Lipoarabinomannan
129