Purification of Proteoglycans by Preparative DEAE Microchromatography

1. The stocks listed in Table 2 are required to generate the DEAE column buffers listed in Table 3. The stocks and DEAE column buffers are stored at room temperature unless stated otherwise. All column buffers are 0.45-^m filtered.

2. Preparative wash buffer. A 50-mL solution of 150 mM NaCl, 0.03% Triton X-100, and 30 ^g/mL BSA is made from Table 2 stocks, then stored at 4°C.

3. Preparative elution buffer. A 50-mL solution of 1 MNaCl, 0.03% Triton X-100, 30 ^g/ mL BSA, and 2.5 ^g/mL glycogen is made from Table 2 stocks, then stored at 4°C.

4. GUTE (100 mL). 4 M guanidine-HCl (Fluka # 50935), 50 mM Tris pH 7.4, 1 mM EDTA. (see Note 4).

5. Chondroitin sulfate (from shark cartilage, Sigma C-4384). Make 2 mL of a 20-mg/mL solution in water, store at -20°C.

6. Column blocking mix. To 1.8 mL of fetal bovine serum add 0.2 mL of the above chondroitin sulfate solution. Spin in a microfuge at maximum speed for 10 min and transfer the supernatant to a clean tube.

7. DEAE microcolumn assembly. A gravity-flow microcolumn is assembled in the barrel of a plastic 1-mL syringe. Onto the tip, fit a Luer adapter with ~4 cm of tubing (trim a "butterfly" catheter; VWR # VT7251). This outflow tract can be plugged by inserting the point of a "yellow" tip (seal the wide end by flame-melting), whereas the top is sealed

Table 2

Stock Solutions for Generating DEAE Column Buffers

Stock

Comments

10 M Urea, 500 mL

1.0 M Na2SO4, 100 mL

20 mg/mL Glycogen, 1 mL

Autoclave sterilize. Autoclave sterilize.

Insoluble at low pH, add NaOH pellets while stirring and monitor pH. Sigma (# C3023) To 80 mL of stirring water, gradually add solid. Cover with Parafilm, stir overnight, adjust volume, 0.45-^m filter. Lot variability see Note 3.

A saturated solution. Complete dissolution requires heating at 37°C. If crystals form upon storage, dissolve by reheating.

Extremely toxic. Wear mask, gloves, and coat when weighing out powder.

Extensive stirring required. Store at 4°C.

RIA grade bovine serum albumin (BSA) (Sigma #

A7888). Reserve aliquots (2 mL) stored at -80°C, working aliquots stored at -20° C.

Boehringer Mannheim (# 901 393)_

with a microstopper (Thomas Scientific, # 8751-K10). A bottom frit is punched out of porous polyethylene (Labconco # 4330115) with a #2 cork bore, then inserted into the barrel. Flush the syringe with water, then add 320 ^L of 50/50 slurry of DEAE Sepharose Fast Flow (Pharmacia #17-0709-01). Adjust to generate a 160-^L matrix bed.

8. Blocking the DEAE column (see Note 5). Drain the column by gravity, then apply 200 ^L of column blocking mix. After 10 min, wash the column by the sequential application of 1 mL of equilibration buffer, three applications of 1 mL of column wash, 1 mL of equilibration buffer, 1 mL of GUTE, and 1 mL of equilibration buffer. Store as plugged columns containing ~1 mL of equilibration buffer.

9. Final column preparation. Before sample application drain the column. Resuspend the bed by forcefully squirting, in rapid succession, two 0.5-mL volumes of equilibration buffer directly at the matrix. Allow the bed to repack by gravity flow, then repeat. Pass through 1 mL of column wash, followed by 1 mL of urea/acid wash. The column is ready for loading (see Subsection 3.2, step 1).

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