Blocking Peptide

A blocking peptide — sometimes called a peptide competitor or neutralising peptide — is the free synthetic peptide matching the immunogen sequence against which an antibody was raised. It is sold together with that antibody for a single reason: to show that a detected signal really originates from the intended epitope.

Running the control

Before the antibody goes onto the blot, plate or tissue section, it is incubated with a molar surplus of the peptide — typically anywhere from several-fold to a hundred-fold, tuned by experiment. A signal that depends on the epitope should disappear or drop sharply; any band that remains is either non-specific or comes from a separate, cross-reacting epitope. Blocked and unblocked conditions have to be run in parallel on one and the same sample, and adding a peptide-only lane is sensible to show that the peptide produces no signal of its own.

Be clear about what this does not prove: it establishes dependence on the epitope, not that the stained protein is the one you think it is. Any unrelated protein carrying the same short linear epitope would respond in exactly the same way. Knockout or knockdown of the gene is still the more rigorous specificity control; peptide blocking is the convenient one.

Related terms and material

polyclonal vs monoclonal · purified IgG · ELISA · native sequence. Reference material: antibodies, peptide libraries. Further reading: antibodies in peptide research.