For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
If you continue without changing your cookie settings, we'll assume you’re happy with this.
Non-specific binding of an antibody to proteins other than the antigen can sometimes occur. This is usually more common with polyclonal antibodies, but can also occur with monoclonals.
To determine which band or staining is specific, an immunizing peptide blocking experiment can be performed. Before proceeding with the staining protocol, the antibody is neutralized (incubated with an excess of peptide that corresponds to the epitope recognized by the antibody). The antibody that is bound to the blocking peptide is no longer available to bind to the epitope present in the protein on the western blot or in the cell.
The neutralized antibody is then used side-by-side with the antibody alone, and the results are compared. By comparing the staining from the blocked antibody versus the antibody alone, you can see which staining is specific; this staining will be absent from the western blot or immunostaining performed with the neutralized antibody.
If more than one band disappears in western blot by peptide/antigen competition, those bands contain the antigenic determinants and could be fragments of the full antigen or a complex containing the antigen.