29. Evaporate to dryness under vacuum with a rotary evaporator.
30. Resuspend LAM in minimum volume of water (<1 mL) and
precipitate overnight at 4
C with 10–20 volumes of ethanol.
31. Centrifuge at 3000 Â g at 4
C, remove the ethanol and
lyophilize.
3.22 Analysis of LAM
by SDS-PAGE
The homogeneity of LAM fractions can be easily and quickly
determined by SDS-PAGE analysis and periodic acid-silver nitrate
staining. LAM and LM migrate as broad bands around 30 and
20 kDa, respectively, according to protein standards (see
Fig. 5) [60].
1. Submit 1 μg of LAM to classical SDS-PAGE using 15%
SDS-polyacrylamide gels.
2. Fix the gel for 20 min by gently rocking in a solution of 50%
methanol, 12% TCA, and 2% CuCl 2 .
3. Remove the fixative and place the gel in a solution of 10%
ethanol and 5% acetic acid (solution A) for 10 min.
4. Remove solution A and add a 0.7% solution of periodic acid in
40% ethanol and 5% acetic acid for 10 min.
5. Wash the gel in solution A for 10 min, then in a solution of 10%
ethanol for 10 min and finally in water for 10 min.
6. Place the gel in 0.1% silver nitrate solution for 10 min.
7. Rinse the gel with water for 30 s, then with a 10% potassium
carbonate solution for 1 min.
kDa
1
2
40LAM
LM
10Fig. 5 SDS-PAGE analysis of M. tuberculosis LAM and LM. Lane 1: Protein MW
standards. Lane 2: LAM and LM fractions from M. tuberculosis H37Rv after
hydrophobic interaction chromatography
140
Marie-Antoinette Lane ´ elle et al.
30. Resuspend LAM in minimum volume of water (<1 mL) and
precipitate overnight at 4
C with 10–20 volumes of ethanol.
31. Centrifuge at 3000 Â g at 4
C, remove the ethanol and
lyophilize.
3.22 Analysis of LAM
by SDS-PAGE
The homogeneity of LAM fractions can be easily and quickly
determined by SDS-PAGE analysis and periodic acid-silver nitrate
staining. LAM and LM migrate as broad bands around 30 and
20 kDa, respectively, according to protein standards (see
Fig. 5) [60].
1. Submit 1 μg of LAM to classical SDS-PAGE using 15%
SDS-polyacrylamide gels.
2. Fix the gel for 20 min by gently rocking in a solution of 50%
methanol, 12% TCA, and 2% CuCl 2 .
3. Remove the fixative and place the gel in a solution of 10%
ethanol and 5% acetic acid (solution A) for 10 min.
4. Remove solution A and add a 0.7% solution of periodic acid in
40% ethanol and 5% acetic acid for 10 min.
5. Wash the gel in solution A for 10 min, then in a solution of 10%
ethanol for 10 min and finally in water for 10 min.
6. Place the gel in 0.1% silver nitrate solution for 10 min.
7. Rinse the gel with water for 30 s, then with a 10% potassium
carbonate solution for 1 min.
kDa
1
2
40LAM
LM
10Fig. 5 SDS-PAGE analysis of M. tuberculosis LAM and LM. Lane 1: Protein MW
standards. Lane 2: LAM and LM fractions from M. tuberculosis H37Rv after
hydrophobic interaction chromatography
140
Marie-Antoinette Lane ´ elle et al.