Recombinant mouse DR5 (aa53-177)-Fc fusion protein (721-DR, R&D) alone (10 µg), or DR5–mTRAIL complex (10 µg each pre-incubated for 1 hr at room temperature), were loaded onto heparin-Sepharose (Cytiva) gravity column (200 µl bed volume). Column was first washed with 2 ml buffer A (25 mM HEPES, pH7.1, 150 mM NaCl), followed by four elution steps (800 µl each) using buffers containing 300 mM, 500 mM, 1 M, and 2 M NaCl, respectively. 30 µl of eluents from each step were resolved on a 4–20% SDS-PAGE gel and the gel was visualized by silver staining.
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