O glycanase

To remove O-linked glycans from proteins, whole cell lysates in RIPA buffer were treated with Neuraminidase (New England Biolabs GmbH, Frankfurt am Main, Germany; Cat. No. P0720S) and Endo-α-N-acetylgalactosaminidase, also known as O-glycanase (New England Biolabs; Cat. No. P0733S), according to the manufacturer's instructions. Therefore, a total protein amount of 90 μg was adjusted with RIPA buffer to a final volume of 45 μl, followed by the addition of 5 μl of 10× Glycoprotein Denaturing Buffer (5% SDS, 0.4 M DTT). Proteins were denatured by heating at 95°C for 10 min. The chilled reaction was supplemented with 7 μl G7 Buffer (0.5 M sodium phosphate, pH 7.5) and 7 μl of 10% NP-40. The reaction was divided in to three equal parts. One third was supplemented with 2 μl ddH2O giving the negative control. The second third was supplemented with 1 μl ddH2O and 1 μl Neuraminidase (50 U/μl). The last third was supplemented with 1 μl Neuraminidase and 1 μl O-glycanase (40,000 U/μl-1). All reactions were incubated at 37°C for 3.5 h. The reactions were stopped by addition of protein sample buffer (Roti-Load 1; Carl Roth, Karlsruhe, Germany, K929.1).