N glycosidase a

N-Glycosidase A (5 mU; Roche Life Science, Indianapolis, IN) was diluted with 100 mM sodium acetate buffer (pH 5.0) to a concentration of 0.25 mU per 16 μl. rhTSG-6 (1.2 μg) was added, the sample volume was increased to 20 μl with the enzyme diluent, and it was incubated at 37°C for 15, 60, and 180 min. Half of the reaction solution was separated by 10% SDS-polyacrylamide electrophoresis and stained with Coomassie Brilliant Blue solution. The remainder was separated by 10% SDS-polyacrylamide electrophoresis and transferred to nitrocellulose (NC) membrane for staining glycoproteins (Pierce Glycoprotein Staining Kit; cat. # 24562; Thermo Scientific, Rockford, IL). In brief, the NC membrane was immersed in 10 ml 3% acetic acid for 10 min and then transferred to 10 ml of oxidizing solution. After 15 min gentle agitation, the membrane was washed with 10 ml of 3% acetic acid solution for 5 min three times. The membrane was soaked in 10 ml of Glycoprotein Staining reagent for 15 min and then transferred to 10 ml of Reducing Solution and gently washed with 3% acetic acid for 5 min three times. To visualize the stained bands, the membrane was washed with ultrapure water. The stained membrane was stored in 3% acetic acid solution.