*4.2. Human Apo D Purification*

Human Apo D (hApo D) was purified from BCF samples provide by the Pathology Unit of the Hospital Universitario Central de Asturias (HUCA). First, a cell fractionation was performed by differential centrifugation. Then, Amicon® Ultra-15, 100 kDa, centrifugal filter units (Z740211, Sigma-Aldrich, St. Louis, MO, USA) were used. The solution containing the protein was flow through two consecutively ion-exchange chromatographic columns (HiTrap® Q Fast Flow, GE Healthcare, Chicago, IL, USA) with 25 mM Tris pH 8.0, followed by a size-exclusion chromatography (HiLoad® 16/60 Superdex® 200 prep grade, GE Healthcare, Chicago, IL, USA ) in 50 mM Tris pH 8.0, 75 mM NaCl. Elution fractions with the protein of interest can be further concentrated using an appropriate 30 kDa cut-off Amicon® centrifuge filter (Z717185, Sigma-Aldrich, St. Louis, MO, USA). The presence of hApo D in these fractions was checked by Western blot, the amount of this apolipoprotein (concentration in the fraction) was quantified and its functionality was tested.
