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Updated: Oct 7, 2026

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis
Published on: February 5, 2014
Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting
1Laboratoire National de Dépistage du Dopage, 143, avenue Roger Salengro, 92290 Châtenay-Malabry, France. f.lasne@wanadoo.fr
Abstract:
"Double-blotting" (DB) was developed to overcome the problem of non-specific 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 first one. The second membrane could then be probed by the secondary antibodies without the risk of non-specific binding. This method was developed for the study of erythropoietin (EPO) in concentrated urine since a strong non-specific binding of biotinylated secondary antibodies to some urinary proteins had been observed using classical IB protocols.
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