Tips and tricks in the preparation of anti-peptide antibodies
Some tips and tricks in the preparation of anti-peptide antibodies reactive to whole proteins are described. An emphasis is made on the preparation of isoform- and conformation-specific antibodies, precautions during the peptide synthesis, application of new approaches to the preparation of peptide-carrier conjugates and thorough choice for carriers and linkers, use of animals for anti-peptide antibody production and specific antibody separation.
Anti-peptide antibodies are directed against a certain protein fragment or against a free peptide (1-6). These antibodies have advantages of a defined specificity, compared to polyclonal and monoclonal antibodies raised against whole proteins. Anti-peptide antibodies are usually employed if: 1) proteins of interest are not available in amounts required for the immunization, but their sequences are known; 2) a protein is toxic and cannot be used for the immunization; 3) a protein is highly conserved, hence its injection may result in an inefficient antibody production or cause autoimmune reactions in immunized animals; 4) protein isoforms with highly similar sequences, splice variants or differently modified forms of the same protein should be distinguished; 5) location or conformation of protein sequence fragments in different conformational states should be compared, with no 3D-structural data available; 6) group-specific antibodies recognizing various proteins with identical or highly similar sequence fragments are required; 7) selective detection of protein-derived peptide fragments in complex mixtures is performed. The latter application is realiz ...