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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 as well. 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.
Blocking peptides and the corresponding antibodies can be found either in the applications section of the peptide datasheet or on the immunogen section of the antibody datasheet.
Materials and reagents
1. Blocking buffer: 5% non-fat dry milk in TBST (NFDM/TBST)
2. Specific antibody blocking peptide
1. Dilute the antibody to 1:100 in 5% NFDM/TBST (1µl in 99µl 5% NFDM/TBST)
Note: optimal concentration of antibody that consistently gives a positive result in your particular protocol may be required
2. Add 5µl (1mg/ml) peptide to 100µl diluted antibody
3. Incubate with agitation overnight at 4ºC
4. Run the western blot. Use the antibody without the the blocking peptide as a control.