The preparation of ferritin-labelled antibodies and other protein-protein conjugates with bis-diazotized benzidine

Abstract

1. 1. A study has been made of the conditions affecting the conjugation of proteins with proteins by bis-diazotized benzidine. Conjugates have been detected by analytical ultracentrifugation, cellulose acetate electrophoresis and immunoelectrophoresis. 2. 2. The optimal conditions have been determined for using bis-diazotized benzidine in the preparation of ferritin-labelled antibodies for use in electron microscopy. 3. 3. The amount of protein-protein conjugate formed was found to depend largely upon the protein concentration and the molar ratio of protein to bis-diazotized benzidine in the reaction mixture. 4. 4. A portion of the conjugates formed at neutral pH was dissociable by treatment with phenolic compounds or by subjection to extremes of pH. The implications of this concerning the mechanism of reaction between bis-diazotized benzidine and protein are discussed.