*5.4. Albumin-Depleted Human Plasma*

To deplete serum albumin from human plasma, we employed Blue Sepharose CL-6B resin affinity chromatography (Sigma-Aldrich, St. Louis, MO, USA; [108,109]). Three independent experiments were carried out with plasma samples from three individuals. The procedure was performed using a microtube with a filter, according to the manufacturer's instructions. In the upper part of the filter microtube, 0.5 mL of resin was added, and steps for resin conditioning, binding and elution, and resin reconstitution were carried out by fractioning the total volume required in 0.5 mL aliquots, added to the top of the microtube, followed by mild manual agitation and centrifugation for 1 min at 150× *g*, with disposal or storage of the eluted solution conducted according to the stage. The following solutions were added: (i) conditioning: 2.5 mL of ultrapure water and 4.0 mL of binding buffer (20 mM HEPES, pH 7.4), with disposal of the eluted solution; (ii) binding: the lower outlet duct of the micro tube containing the resin was closed, and 40 μL of plasma diluted in 310 μL of binding buffer was loaded. The tube was kept at room temperature for 30 min under agitation. After centrifugation, the eluted material, corresponding to the plasma depleted of albumin, was stored. For the elution of proteins bound to the resin (eluate), 4 mL of 2 M NaCl were added to the resin, and the eluted solution was stored. For resin reconstitution, 1 mL of 6 M guanidine hydrochloride and 4 mL of binding buffer were applied to the resin, and the eluted solution was discarded. Albumin-depleted plasma samples were designated as P(Alb-D).
