Native page bis tris gels of gradient 4 to 16 t

Collected CN-GELFrEE fractions were analyzed via native PAGE bis-tris gels of gradient 4 to 16%T (Thermo Fisher Scientific, Waltham, MA). NativeMark Unstained Protein Standard (Thermo Fisher Scientific, Waltham, MA) was used as the molecular weight ladder. A 10 μL aliquot of each fraction and a 10 μL aliquot of a 1/500 dilution of the pooled serum sample in 100 mM ammonium bicarbonate pH 7.4 buffer were each mixed with 4 μL of the same glycerol-Ponceau S loading dye described previously. Each gel was run at room temperature at a constant 120 V. For fractions of the 8 cm CN-GELFrEE, each was run on two separate native gels, one for silver staining (following a previously described protocol 22 ) and one for anti-APOA1 Western blotting (described below). For the 25 cm CN-GELFrEE only the Western blotting analysis was performed and only for fractions that had previously been shown to contain APOA1 by LC-MS (see below).