Avoiding Nonspecific Binding of Secondary Antibodies in Immunoblotting by Double-Blotting

False positives occur in immunoblotting methods due to nonspecific binding between proteins transferred to a membrane and unrelated secondary antibodies. Some methods are available to reduce this adsorption, but they may be effective in specific applications and not work in other cases. Francoise Lasne developed “double-blotting” (DB) to overcome this problem. This method involved inserting a second blotting step between the blot membrane’s normal probings with the primary antibody and the secondary antibodies. This step assures the probing’s specificity with the secondary antibody by isolating the primary antibody from the interfering proteins. Lasne developed this technique to study erythropoietin (EPO) in concentrated urine to overcome the robust nonspecific binding of biotinylated secondary antibodies to certain urinary proteins seen with traditional immunoblotting procedures. This method could be of use in studying proteins present in minuscule quantities in complex biological samples.