Preparation of glycosaminoglycans

 Homogenize a tissue in ice-cold acetone.

 Desiccate the acetone powder.

 Suspend the dried material (25 g) in distilled water (40 mL).

 Inactivate contaminating glycosidases by boiling at 100˚C for 10 min.

 Digest the sample with actinase E (0.25 g) in a total volume of 65 mL of 0.1 M borate buffer, pH 8.0, containing 10 mM CaCl₂ at 65˚C for 2 days.

 Treat the digest with 5% trichloroacetic acid at 4˚C for 1 h to precipitate residual proteins and peptides.

 Centrifuge at 3,500 rpm for 10 min to remove the precipitate.

 Remove trichloroacetic acid by extraction with an equivalent volume of diethylether 5 times.

 Neutralize the aqueous phase with 1 M Na₂CO₃.

 Precipitate the crude GAG with 80% ethanol containing 1% sodium acetate at 4˚C overnight.

 Centrifuge at 3,000 rpm for 10 min.

 Reconstitute the precipitate in distilled water.

 Load the sample onto a prepacked disposable PD-10 column equilibrated with H₂O to be desalted.

 Lyophilize the flow-through fraction by Speed Vac.

 Quantify the yield of GAGs by the carbazole reaction.

 Load the crude GAG fraction (2.5 mg as GAG) on an Accell QMA Plus cartridge pre-equilibrated with 0.05 M phosphate buffer, pH 6.0, containing 0.15 M NaCl.

 Wash the column with 2 mL of the equilibration buffer.

 Elute GAGs from the column with 3 mL each of 0.05 M phosphate buffer, pH 6.0, containing 0.5 and 2.0 M NaCl.

 Dialyze the eluates against water using Microcon^® YM-10 and concentrate them by Speed Vac.

 Lyophilize samples (cells/small amount of tissues) over 16 h.

 Homogenize the samples (up to 10⁷ cells/up to 20 mg of lyophilized tissue) in 1.0 mL of acetone cooled on ice. Stand for 15 min on ice, then centrifuge at 2,300 × g for 15 min at 4℃. Wash the precipitate with 1.0 mL of ice-cold acetone and dry under vacuum.

 Extract samples in 1.0 mL of GAG extraction solution (0.5% SDS, 0.1 M NaOH, and 0.8% NaBH₄) for 16 h at room temperature with constant stirring.

 Neutralize samples with 200 μL of 1.0 M sodium acetate and 300 μL of 1.0 M HCl and centrifuge at 2,300 × g for 10 min at 4℃. Remove particulate matter from the supernatant with a 300-μm pore disposable filter column.

 Add 200 μL of 1.0 M HCl to the filtrate and remove insoluble materials by centrifugation at 2,300 × g for 10 min at 4˚C. Add 7 mL of ethanol saturated with sodium acetate to the supernatant and allow GAGs to precipitate for 16 h at 4˚C.

 Centrifuge at 2,300 × g for 15 min at 4℃ and remove the supernatant. Wash the precipitate once with 1 mL of 80% ethanol and once with 1 mL of ethanol. Dry the precipitate under vacuum. At this stage, the crude GAG pellets can be stored at −20℃ until needed.

 Dissolve the crude GAG pellets in 250 μL of water.

 Add 50 μL of 0.3 M sodium phosphate buffer, pH 6.0, to the crude GAG solution.

 Filter the mixture through an Ultrafree MC (Durapore, 0.45 μm).

 Add the filtrate to the Ultrafree MC DEAE insert pre-equilibrated with loading buffer (50 mM sodium phosphate buffer, pH 6.0) and spin at 5,000 × g for 1 min. Pass the sample over the membrane twice.

 Add 400 μL of loading buffer to the insert and spin at 5,000 × g for 1 min.

 Transfer the insert to a new microcentrifuge tube. Add 100 μL of elution buffer (50 mM sodium phosphate buffer, pH 6.0, containing 1.0 M NaCl) to the insert and spin at 5,000 × g for 1 min. Repeat 3 times. This fraction is the eluate.

 Add the eluate to Ultrafree MC (Biomax 5) and spin until the retentate is about 30 μL.

 Add 50 μL of distilled water to the retentate and spin. Repeat four times.

 Remove the retentate to a new micro tube and rinse the membrane four times with 20 μL of distilled water. Add the washes to the retentate.

 Dry the sample by Speed Vac.