Typically, 10 mL of human plasma or serum was diluted 20-fold with the manufacturers equilibration buffer, filtered through a 0.22 mm microcentrifuge filtration tube, and loaded onto the antibody resin in a microcentrifuge spin column. The sample was either passed into the column at slow speed (1.5 min, 100 x g, RT) or incubated at 47C for 15 min. In both cases, the column was washed twice with the equilibration buffer and centrifuged (2.5 min, 100 x g) to collect the total unbound plus wash fractions. The bound fraction was collected with the manufacturer's elu-tion buffer and neutralized, and both the flow-through fraction containing unbound proteins and the bound fractions were concentrated with a 5K MWCO spin concentrator for immediate analysis or aliquoted and stored at —807C for future use.
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