Assaying for the presence of glycanated glypican-AP fusion protein in the cell media:
AP activity in the media was assayed by adding an equal volume of the phosphatase substrate p-nitrophenyl phosphate (PNP) from Sigma Inc. (St. Louis, MO; made in 2M diethanolamine and 1 mM MgCl2) to 50 µl of cell medium and then, incubated at 37ºC for 5 min. AP activity is signaled by a change in the color of the solution. The type of GAG chains presentHS or chondroitin sulfatewas determined by treating the cell media with heparitinase, chondroitinase ABC, or both, and separating the digested products on a 1% low melting point agarose gel made in 50 mM Tris-Cl (pH 8) and 1 mM MgCl2. The AP-containing proteins on the gel were detected with the addition of an AP substrate (NBT/BCIP), made from Sigma Fast tablets (Sigma). The solution turns blue in the presence of AP. The cos-7 cell medium was also analyzed by Western blotting, where the protein was probed with a mouse anti-AP antibody according to the ECL Western blotting protocol (Life Science).
Purifying the fusion proteins with diethylaminoethyl (DEAE) chromatography:
A binding column, with a volume equal to 30% of that of the protein sample to be purified, was made using DEAE Sephacel beads (Pharmacia Biotech Inc., Piscataway, NJ) and equilibrated with 10 column volumes of 50 mM Tris-Cl buffer (pH 8) containing 150 mM NaCl. After 30 to 40 ml of cell medium passed through, the column was washed with two column volumes of 50 mM Tris-Cl (pH 8) containing 150 mM NaCl, followed with the same buffer containing 250 mM NaCl. The column was eluted with two column volumes of 750 mM NaCl made in the same buffer, and collected in 1.5 to 1.75 ml fractions. The fractions with the highest amount of AP activity, as determined by a visual inspection of their color (using AP substrate PNP) after 5 to 10 min of incubation at 37ºC, were pooled. Protease inhibitors (250 µg/ml NEM, 1 µg/ml Pepstatin A, and 0.5 mM PMSF) were added to the combined fractions, which were then dialyzed against ACE buffer (see below) and stored at -80ºC.
Measuring binding by affinity co-electrophoresis:
The binding affinities of the glypican fusion protein were determined by ACE (Herndon and Lander 1997; Figure 2). ACE gels were developed by incubation with the AP substrate NBT/BCIP at 37ºC for several hours or overnight, analyzed using the software ImageQuant (Molecular Dynamics, Inc. Sunnyvale, CA) and quantified essentially as described (San Antonio et al. 1993), except that a colorimetric rather than radioactivity-based method was used to determine mobility. For each lane, relative AP-staining intensity as a function of the distance from the top of the lane was measured