Sometimes non-specific binding of an antibody to proteins other than the antigen can take place. This is typically more common with polyclonal antibodies, but can also happen with monoclonals as well.
To establish which band or staining is specific, an immunizing peptide blocking experiment can be undertaken. Before continuing with the staining protocol, the antibody is neutralized (incubated with extra peptide that corresponds to the epitope detected by the antibody).
The antibody that is bound to the blocking peptide is not available anymore to bind to the epitope found in the protein on the Western blot or in the cell. The neutralized antibody is then used side-by-side with just the antibody, and the results are compared.
Users can see which staining is specific by comparing the staining from the blocked antibody versus the antibody alone. This staining will be absent from the immunostaining or Western blot done using the neutralized antibody.
Blocking peptides and the corresponding antibodies can be seen either in the applications section of the peptide datasheet or on the immunogen section of the antibody datasheet.
Materials and Reagents
- Blocking buffer: 5% non-fat dry milk in TBST (NFDM/TBST)
- Specific antibody blocking peptide
- Dilute the antibody to a ratio of 1:100 in 5% NFDM/TBST (1 µl in 99 µl 5% NFDM/TBST)
Note: Optimal concentration of antibody that steadily gives a positive result in a specific protocol may be required.
- Add 5 µl (1 mg/ml) peptide to 100 µl diluted antibody
- Incubate with agitation overnight at 4 °C
- Run the western blot. The antibody can be used without the blocking peptide as a control.
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