Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting procedures

"Double-blotting" (DB) was developed to overcome the problem of non-specifi c binding of secondary antibodies in immunoblotting (IB). After it had been probed by the primary antibody, the membrane with the blotted proteins was assembled with a second blank membrane and submitted to a second blotting under acidic conditions. The primary antibody molecules were thus desorbed from their corresponding antigen and transferred onto the second membrane, whereas the antigen and the interfering proteins remained bound to the firs t one. The second membrane could then be probed by the secondary antibodies without the risk of non-specific binding. This method was developed for th e study of erythropoietin (EPO) in concentrated urine since a strong non-sp ecific binding of biotinylated secondary antibodies to some urinary protein s had been observed using classical IB protocols. (C) 2001 Elsevier Science B.V. All rights reserved.