Preparation of a soluble form of CD44 from insect cell culture medium. (A) Ammonium sulfate is responsible for retention of soluble CD44 on PVDF filters. 22 μg CD44 prepared as indicated in Methods were dissolved in 0.3 ml 0.1 M Tris-HCl, pH8 and filtered through Millex-GV low protein binding Durapore, 0.22 μm pore size (Millipore SLGV033RS) in Tris buffer alone or with 3 M ammonium sulfate. The ultrafiltrates of ammonium-sulfate containing solutions were practically free of immunoreactive CD44, measured as indicated in Methods. Values are mean ± SEM (n = 4). (B) Reverse phase HPLC of the soluble ammonium sulfate fraction of cell culture supernatants. The supernatants of TN cultures expressing CD44 were processed as indicated in Methods and applied to a Vydac C4 semi-preparative column. (B1) Acetonitrile gradient, 0% to 72% (v/v) acetonitrile in 0.1% (v/v) TFA for 50 min, 2 ml/min. (B2) After centrifugation of the proteins precipitated with ammonium sulfate, the supernatant was desalted and concentrated by ultrafiltration (Centricon-20, cut-off 10 kDa, Millipore) and applied to the column. (B3) Instead of ultrafiltration, the supernatant was filtered through PVDF membranes (type GV, 0.22 μm). The proteins on the filter were extracted and applied to the column. The gray bars represent the density of the immunoreactive fractions using an anti-CD44 antibody. (C) PNGase digestion of recombinant soluble CD44. 120 ng CD44 were applied onto PAGE-SDS gels (10% acrylamide) and separated by electrophoresis before (-) and after (+) 1 h digestion with PNGase F. M = molecular weight markers. The gels were stained with coomassie blue.