Peptide Blocking - A method to confirm antibody specificity.
Background: The specific binding of an antibody to its target epitope is required to generate accurate expression data. Binding of antibody to non-target antigens, also known as non-specific binding, increases background signal and complicates data interpretation. In western blot analysis, non-specific binding is often easily identified by the presence of multiple bands. However, identification of non-specific antibody binding can be more difficult in immunohistochemistry, ELISA, flow cytometry, ChIP, and other antibody based applications.
What is a blocking peptide? Blocking peptides are peptides comprised of the amino acid sequence corresponding to the antibody epitope (specific piece of antigen recognized by the antibody). Blocking peptides will bind specifically to the target antibody, preventing subsequent antibody binding to target epitope.
How does blocking peptide work? The high affinity of an antibody for its epitope can be exploited to confirm binding specificity. Pre-incubation of an antibody with sufficient peptide occupies all antibody binding sites, preventing subsequent target protein binding in sample.
How do I know if my antibody is specific after running a blocking peptide assay? To confirm antibody specificity, compare the strength of signal between the samples stained with control and blocked antibody. If the antibody is specific for the target epitope, experimental samples incubated with blocked antibody will demonstrate less robust signal than the control antibody (antibody alone, not blocked).
What should my peptide blocking data look like in western blot? If the antibody is incubated with excess peptide and all binding sites are occupied (blocked), then the sample incubated with blocked antibody should demonstrate no signal.
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Example Data Using Peptides & Proteins
Western Blot - Preabsorption/ Incubation with Blocking Peptide:
Immunohistochemistry - Preabsorption/ Incubation with Blocking Peptide:
Western Blot - Peptide as Positive Control in Western Blot:
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