Suppression of nonspecific binding of avidin-biotin complex (ABC) to proteins electroblotted to nitrocellulose paper.
Nitrocellulose blots of cell extracts reacted in sequence with biotinylated lectins and horseradish peroxidase-labeled avidin-biotin complex (ABC) often show considerable nonspecific staining of protein bands. Experiments were performed to determine which of the components of the ABC were responsible for this and whether or not the nature and ionic strength of the buffer used could alter this binding. Furthermore, as powdered non-fat milk has been proposed as a possible blocking agent for nonspecific binding of ABC, we sought to determine if it would adequately block that binding in our system. The initial experiments showed that nonspecific binding of ABC to proteins transferred to nitrocellulose membranes was due to the avidin component of the ABC; little, if any, binding was seen if biotin alone was incubated with these blots. The spurious binding was shown to be primarily due to the high affinity of avidin to proteins electroblotted to nitrocellulose, when incubated in low-salt buffers. Low-fat milk added to the buffer reduced overall nonspecific reactivity but produced additional artifacts in the form of bands that were not seen in other preparations. Nonspecific avidin binding to proteins transferred to nitrocellulose can therefore be effectively reduced by adding extra salt to buffers, whereas the addition of non-fat dry milk does not seem suitable for this purpose.