Protocol for Performing Immunizing Peptide Blocking Experiment

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

  1. Blocking buffer: 5% non-fat dry milk in TBST (NFDM/TBST)
  2. Specific antibody blocking peptide
  3. Microtubes

Method

  1. 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.

  1. Add 5 µl (1 mg/ml) peptide to 100 µl diluted antibody
  2. Incubate with agitation overnight at 4 °C
  3. Run the western blot. The antibody can be used without the blocking peptide as a control.

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: Jun 3, 2019 at 10:24 AM

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