Ecl immunoblotting detection reagent

Serum albumin proteins were purified as described previously;^{21 ,22 (link)} mouse serum (50 μL) was diluted with 50 mM potassium phosphate buffer, pH 7.4 (1 mL), prior to affinity chromatography using HiTrap Blue affinity resin. The concentration of the eluted protein was estimated by measuring UV absorption at 280 nm.^{23 (link)} The purity of the eluted protein was assessed via sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, with authentic human and mouse serum albumin proteins (Sigma-Aldrich) as controls. Immunoblot analysis was performed as described^{24 (link)} with use of a goat anti-human albumin antibody that does not cross-react with mouse albumin (Bethyl Laboratories), at a 1:3,000 dilution. Immunoreactive proteins were visualized with use of an Amersham ECL immunoblotting detection reagent and the luminescent images were recorded by a Gel-Doc imaging system (Bio-Rad).