8. Develop the gel in a 2% potassium carbonate solution containing 0.1% formaldehyde until the gold-colored bands appear
and the desired color intensity is reached; stop the reaction by
placing the gel in a 10% ethanol and 5% acetic acid solution for
2 min.
The gel can then be stored in water.
3.23 Analysis of LAM
Glycosyl Composition
LAM is composed of mannosyl and arabinosyl units [60–62].
1. Hydrolyze LAM (100 μg) in the presence of mannoheptose
(100 nmol) as an internal standard using 200 μL of 2 M TFA at
110
C for 2 h in glass vials with Teflon seals.
2. Allow the solution to cooldown and evaporate to dryness
under air stream at 40
C or using a SpeedVac.
3. Add eight drops of pyridine, four drops of hexamethyldisilazane, and two drops of trimethylchlorosilane. Keep for 15 min
at room temperature and evaporate to dryness under an air
stream at 40
C.
4. Resuspend the trimethylsylilated monosaccharides in 200 μL of
cyclohexane and analyze by GC or GC/MS. Peak attribution
can be made by a simple comparison with commercial arabinose and mannose standards submitted to the same protocol
(see Note 21).
3.24 Analysis of LAM
Fatty Acyl Composition
LAM contains mainly palmitic (C 16 ) and tuberculostearic (C 19 )
acids with traces of stearic acid (C 18 ).
1. Deacylate LAM (200 μg) in the presence of pentadecanoic acid
(8 nmol) as an internal standard by saponification using 400 μL
of 1 M NaOH at 110
C for 2 h in glass vials with Teflon seals.
2. Neutralize the reaction mixture with HCl and extract three
times the liberated fatty acids with 400 μL of chloroform.
Combine the chloroform extracts and evaporate to dryness
under an air stream at 40
C.
3. Add three drops of a commercial solution of BF 3 in methanol.
Incubate for 5 min at 60
C and stop the reaction by the
addition of 400 μL of deionized water.
4. Extract three times the fatty acid methyl esters with 400 μL of
chloroform. Combine the chloroform extracts and evaporate
to dryness under an air stream at 40
C.
5. Resuspend the fatty acid methyl esters in 200 μL cyclohexane
and analyze by GC or GC/MS.
Lipid and Lipoarabinomannan
141
and the desired color intensity is reached; stop the reaction by
placing the gel in a 10% ethanol and 5% acetic acid solution for
2 min.
The gel can then be stored in water.
3.23 Analysis of LAM
Glycosyl Composition
LAM is composed of mannosyl and arabinosyl units [60–62].
1. Hydrolyze LAM (100 μg) in the presence of mannoheptose
(100 nmol) as an internal standard using 200 μL of 2 M TFA at
110
C for 2 h in glass vials with Teflon seals.
2. Allow the solution to cooldown and evaporate to dryness
under air stream at 40
C or using a SpeedVac.
3. Add eight drops of pyridine, four drops of hexamethyldisilazane, and two drops of trimethylchlorosilane. Keep for 15 min
at room temperature and evaporate to dryness under an air
stream at 40
C.
4. Resuspend the trimethylsylilated monosaccharides in 200 μL of
cyclohexane and analyze by GC or GC/MS. Peak attribution
can be made by a simple comparison with commercial arabinose and mannose standards submitted to the same protocol
(see Note 21).
3.24 Analysis of LAM
Fatty Acyl Composition
LAM contains mainly palmitic (C 16 ) and tuberculostearic (C 19 )
acids with traces of stearic acid (C 18 ).
1. Deacylate LAM (200 μg) in the presence of pentadecanoic acid
(8 nmol) as an internal standard by saponification using 400 μL
of 1 M NaOH at 110
C for 2 h in glass vials with Teflon seals.
2. Neutralize the reaction mixture with HCl and extract three
times the liberated fatty acids with 400 μL of chloroform.
Combine the chloroform extracts and evaporate to dryness
under an air stream at 40
C.
3. Add three drops of a commercial solution of BF 3 in methanol.
Incubate for 5 min at 60
C and stop the reaction by the
addition of 400 μL of deionized water.
4. Extract three times the fatty acid methyl esters with 400 μL of
chloroform. Combine the chloroform extracts and evaporate
to dryness under an air stream at 40
C.
5. Resuspend the fatty acid methyl esters in 200 μL cyclohexane
and analyze by GC or GC/MS.
Lipid and Lipoarabinomannan
141