2.20
1 NMR of MAME
1. Apparatus 600-MHz Bruker Avance III spectrometer
equipped with a TCI cryoprobe.
2. Topspin software.
3. Highly purified solvent devoid of the nuclei measured:
Deutero-chloroform
(CDCl 3 )
and
deutero-methanol
(CD 3 OD) (see Note 2).
4. NMR 5-mm-diameter glass tube.
5. Pasteur pipettes.
2.21 Purification of
Lipoarabinomannan
(LAM)
1. Glass vessel (see Note 5).
2. Probe sonicator.
3. Rotary vacuum evaporator (see Note 3).
4. Refrigerated centrifuge.
5. SpeedVac.
6. Peristaltic pump.
7. Fraction collector.
8. Magnetic stirrer.
9. Dialysis MWCO 6-8000 tubing.
10. Octyl Sepharose CL-4B.
11. Bio-Gel
® P-100 gel Medium or Sephacryl
® S-200 HR.
12. HPLC-grade ethanol, propan-1-ol, phenol, methanol, chloroform, cyclohexane (see Note 2).
13. PBS.
14. Tris–HCl buffer: 20 mM Tris–HCl, 1 mM MgCl 2 , pH 7.5.
15. Phosphate buffer: 50 mM sodium phosphate, pH 7.
16. Sodium deoxycholate buffer: 0.2 M NaCl, 0.25% sodium
deoxycholate (w/v), 1 mM EDTA, 10 mM Tris, pH 8.
17. Ammonium acetate.
18. DNAse.
19. RNAse.
20. α-Amylase.
21. Chymotrypsin.
22. Trypsin.
2.22 Analysis of LAM
by SDS-PAGE
1. 15% SDS-polyacrylamide gels.
2. Trichloroacetic acid (TCA).
3. Periodic acid.
4. Acetic acid.
5. CuCl 2 .
122
Marie-Antoinette Lane ´ elle et al.
1 NMR of MAME
1. Apparatus 600-MHz Bruker Avance III spectrometer
equipped with a TCI cryoprobe.
2. Topspin software.
3. Highly purified solvent devoid of the nuclei measured:
Deutero-chloroform
(CDCl 3 )
and
deutero-methanol
(CD 3 OD) (see Note 2).
4. NMR 5-mm-diameter glass tube.
5. Pasteur pipettes.
2.21 Purification of
Lipoarabinomannan
(LAM)
1. Glass vessel (see Note 5).
2. Probe sonicator.
3. Rotary vacuum evaporator (see Note 3).
4. Refrigerated centrifuge.
5. SpeedVac.
6. Peristaltic pump.
7. Fraction collector.
8. Magnetic stirrer.
9. Dialysis MWCO 6-8000 tubing.
10. Octyl Sepharose CL-4B.
11. Bio-Gel
® P-100 gel Medium or Sephacryl
® S-200 HR.
12. HPLC-grade ethanol, propan-1-ol, phenol, methanol, chloroform, cyclohexane (see Note 2).
13. PBS.
14. Tris–HCl buffer: 20 mM Tris–HCl, 1 mM MgCl 2 , pH 7.5.
15. Phosphate buffer: 50 mM sodium phosphate, pH 7.
16. Sodium deoxycholate buffer: 0.2 M NaCl, 0.25% sodium
deoxycholate (w/v), 1 mM EDTA, 10 mM Tris, pH 8.
17. Ammonium acetate.
18. DNAse.
19. RNAse.
20. α-Amylase.
21. Chymotrypsin.
22. Trypsin.
2.22 Analysis of LAM
by SDS-PAGE
1. 15% SDS-polyacrylamide gels.
2. Trichloroacetic acid (TCA).
3. Periodic acid.
4. Acetic acid.
5. CuCl 2 .
122
Marie-Antoinette Lane ´ elle et al.