11. Back-extract the phenol phase with PBS as described above.
12. Combine the aqueous phases containing lipoglycans.
13. Dialyze against water (MWCO 6-8000) and evaporate to dryness under vacuum with a rotary evaporator (see Note 3).
14. Suspend the above dried extract in either Tris–HCl or phosphate buffer, according to enzyme used, and submit to sequential enzymatic digestions by DNAse, RNAse (both in Tris–HCl
buffer), α-amylase, chymotrypsin, and trypsin (all three in
phosphate buffer) for 8 h each enzyme using an enzyme/
product ratio of 1% (w/w).
15. Dialyze against deionized water (MWCO 6-8000) and evaporate to dryness under vacuum with a rotary evaporator or using
a SpeedVac (depending on the volume to be evaporated).
16. Dissolve the dried sample in a minimum volume of a solution
of 15% propan-1-ol in 50 mM ammonium acetate.
17. Load the sample onto an Octyl Sepharose CL-4B column
previously equilibrated with the same solution (1 mL of phase
is needed for 1 mg of sample) [58].
18. Elute the column at 5 mL/h with three column volumes of the
same solution, enabling the removal of hydrophilic
compounds.
19. Elute the column with three column volumes of 50% propan1-ol in 50 mM ammonium acetate.
20. Concentrate the latter eluent to less than 1 mL and precipitate
lipoglycans overnight at 4
C with 10–20 volumes of ethanol.
21. Centrifuge at 3000 Â g at 4
C.
22. Remove the ethanol and dry lipoglycans by freeze-drying or
using a SpeedVac.
23. Resuspend lipoglycans in sodium deoxycholate buffer to a final
concentration of 200 mg/mL, incubate for 48 h at 37
C.
24. Load onto a Bio-Gel P-100 or Sephacryl S-200 HR column
(50 Â 2.5 cm) previously equilibrated with the same buffer
[50, 59].
25. Elute with the buffer at a flow rate of 5 mL/h.
26. Collect 1.25 mL fractions and determine the point of elution
of LAM and LM by running 10 μL of each fraction by
SDS-PAGE (see Subheading 3.22).
27. Pool separately fractions containing purified LAM and fractions containing purified LM.
28. Dialyze (MWCO 6-8000) extensively the pooled fractions for
2 days against 0.2 M NaCl, 1 mM EDTA, and 10 mM Tris,
pH 8, to remove sodium deoxycholate and then against deionized water for 2 days.
Lipid and Lipoarabinomannan
139
12. Combine the aqueous phases containing lipoglycans.
13. Dialyze against water (MWCO 6-8000) and evaporate to dryness under vacuum with a rotary evaporator (see Note 3).
14. Suspend the above dried extract in either Tris–HCl or phosphate buffer, according to enzyme used, and submit to sequential enzymatic digestions by DNAse, RNAse (both in Tris–HCl
buffer), α-amylase, chymotrypsin, and trypsin (all three in
phosphate buffer) for 8 h each enzyme using an enzyme/
product ratio of 1% (w/w).
15. Dialyze against deionized water (MWCO 6-8000) and evaporate to dryness under vacuum with a rotary evaporator or using
a SpeedVac (depending on the volume to be evaporated).
16. Dissolve the dried sample in a minimum volume of a solution
of 15% propan-1-ol in 50 mM ammonium acetate.
17. Load the sample onto an Octyl Sepharose CL-4B column
previously equilibrated with the same solution (1 mL of phase
is needed for 1 mg of sample) [58].
18. Elute the column at 5 mL/h with three column volumes of the
same solution, enabling the removal of hydrophilic
compounds.
19. Elute the column with three column volumes of 50% propan1-ol in 50 mM ammonium acetate.
20. Concentrate the latter eluent to less than 1 mL and precipitate
lipoglycans overnight at 4
C with 10–20 volumes of ethanol.
21. Centrifuge at 3000 Â g at 4
C.
22. Remove the ethanol and dry lipoglycans by freeze-drying or
using a SpeedVac.
23. Resuspend lipoglycans in sodium deoxycholate buffer to a final
concentration of 200 mg/mL, incubate for 48 h at 37
C.
24. Load onto a Bio-Gel P-100 or Sephacryl S-200 HR column
(50 Â 2.5 cm) previously equilibrated with the same buffer
[50, 59].
25. Elute with the buffer at a flow rate of 5 mL/h.
26. Collect 1.25 mL fractions and determine the point of elution
of LAM and LM by running 10 μL of each fraction by
SDS-PAGE (see Subheading 3.22).
27. Pool separately fractions containing purified LAM and fractions containing purified LM.
28. Dialyze (MWCO 6-8000) extensively the pooled fractions for
2 days against 0.2 M NaCl, 1 mM EDTA, and 10 mM Tris,
pH 8, to remove sodium deoxycholate and then against deionized water for 2 days.
Lipid and Lipoarabinomannan
139