DETECTION OF DNA-BINDING PROTEINS BY COUNTERFLOW ISOTACHOPHORESIS ON NITROCELLULOSE MEMBRANES .1. ANTIBODIES TO DNA AND TO ITS ADDUCTS

A powerful electroosmotic flow arises during isotachophoresis on a nit rocellulose membrane in a system of 0.06 M Tris- HCl pH 6.7, and 0.012 M Tris-alanine, pH 8.6, as the leading and terminating buffer solutio ns, respectively. This flow opposes the migration of the Cl-/-alanine( -) boundary and stops it. The rate of counterflow exceeds by far the m igration rate of any organic anions, including negatively charged prot eins. Native or denatured DNA or its adducts were fixed on the nitroce llulose membrane, which was blocked with milk proteins. DNA-binding pr oteins, namely anti-DNA antibodies, followed by peroxidase-conjugated anti-IgG, were introduced into the counterflow, which carried them con secutively to the DNA. Thus, multistep binding and washing was perform ed automatically This technique allowed us to reveal the antibodies to ds-, ss-, BrDU-, Z-, and transplatinum-modified DNA, as well as strep tavidin-peroxidase binding to biotinylated DNA.