Factors affecting efficiency of colloidal gold staining: pH-dependent stability of protein-gold conjugates.

Abstract

The preparation of stable protein-gold conjugates is important in the quantitative colloidal gold staining. The protein adsorption onto colloidal gold particles and pH-dependent stability of protein-gold conjugates were examined using albumin and fibrinogen as model proteins. Albumin stabilized gold sols at pH values higher than 5.0. The amount of albumin necessary to stabilize gold sols was lowest at pH 5.6 and slightly increased as the pH of the adsorption medium was increased. Albumin was also able to stabilize gold particles even at acidic pH values ranging from 3.5 to 4.5, if a sufficient amount of albumin was added. Stable fibrinogen-gold conjugates were obtained at pH values above 6.5. Unlike albumin, the same amount of fibrinogen was able to stabilize gold sols as pH was increased from 7 to 10. The stability of the prepared protein-gold conjugates was very sensitive to the pH of the storing medium. The protein-gold conjugates were least stable at the isoelectric pH of the protein. The results suggest that protein-gold conjugates should be prepared at pH where conjugates are most stable, instead of pH close to the isoelectric point.