For removal ofthe top six most abundant proteins, plasma or serum was applied to either a single MARS HPLC column (Agilent Technologies, Wilmington, DE, USA) (4.6 mm x 50 mm) or a two-column configuration where the 50 mm column was connected in tandem to a second, longer 4.6 mm x 100 mm column for a total column length of 150 mm. These HPLC columns contain polyclonal antibodies to human albumin, transferrin, haptoglobin, a-1-antitrypsin, IgG, and IgA. Typically, when the two-column configuration was used, 40 mL ofhuman plasma or serum was diluted fivefold with the manufacturer's equilibration buffer, filtered through a 0.22 mm microcentrifuge filter tube, and injected onto the antibody column. The flow-through fractions containing unbound proteins from sequential injections were collected, pooled, and concentrated using a 5K molecular weight cutoff (MWCO) spin concentrator. The concentrated pool was either used immediately or aliquoted and stored at —80°C for future use. Affinity-bound major proteins were eluted with the manufacturer's elution buffer, neutralized with 1 m NaOH, and pooled for analysis on 1- and 2-D gels.
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