Generating antibodies

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Drawing of an antibody - heavy chains red, light chains yellow.

You are hot in pursuit of an interesting protein but there are no commercial antibodies or they are bad. It's time to think about raising your own antibodies against the protein. It will be a time and money consuming undertaking but once you developed a specific antibody or set of antibodies, you will be in a unique position to generate interesting data.

Early decisions

Type of antibody

polyclonal (a mix of antibodies with different specificities)
  • faster to generate
  • less specific
  • cheaper than monoclonals
monoclonal (many copies of the same antibody)
  • slower to generate
  • more specific
  • after initial setup no animals required; unlimited production from immortal cell line
  • antigen can be impure; subsequent selection of hybridoma clones
  • more expensive than polyclonals

Source organism

Common animals for raising antibodies include rabbit, goat, mouse (required for monoclonal antibodies), and even llamas [1]. The choice depends on which antibodies will be used in conjunction with antibody to be generated and which type of antibody is desired.

Type of antigen to use

full-length protein
  • likely to produce a conformational epitope useful for recognition of folded protein
  • purchase or purification of full-length protein typically more expensive/time consuming
  • full-length protein may not be availalbe and purification may prove difficult
  • natural selection of epitope but epitope location initially unknown
  • epitope may not be specific to protein
  • likely to produce a linear epitope useful for recognition of primary sequence as in immunoblots
  • cheaper, less effort (more readily available instruments)
  • antigen can typically be synthesised without problems
  • known epitope
  • but possibly low antigenicity

Peptide selection

Example of a protein's hydrophobicity according to 2 different scales. Made with R. Bowen's tool.

If you decide to raise antibodies against a peptide or several peptides from the target protein, you will be faced with the question which region to choose. The gist is, you want to pick a 15-20 residue peptide that is on the surface of the protein, i.e. has a high hydrophilicity. Another common approach is to pick sequences from the ends of the protein, since they are typically exposed. Avoid cysteines if possible, since peptides will often be coupled via Cys residues to carriers and since disulphide bridges alter the shape and thus the recognition by antibodies. Still, peptide selection is trial and error.

Online tools

Sequence plots

  • ExPASy's classic ProtScale tool allows you to check a protein sequence against various amino acid scales including several for hydrophilicity/-phobicity
  • pepwindow hydrophobicity tool from the Institute Pasteur (default Kyte-Doolittle)
  • Hydrophobicity tool by Colorado State Uni - well documented but x scale not sufficiently subdivided


Boosting the immune reaction

A small peptide is often not enough to elicit sufficient antibody production. Several methods, also in combination, can be used to stimulated the immune system.

  • Multimers of the peptide often increase antibody synthesis. Multimers can be made by linking several copies of the peptide as branches to a carrier peptide chain.
  • Adjuvants are mixtures co-injected with the antigen to stimulate the immune response. Complete Freund's adjuvant (CFA), typically made from inactivated mycobacteria, is a common choice. However, newer adjuvants like Ribi's adjuvent and Titermax are safer for both scientist and animal. [2] See also adjuvant in the wikipedia.


  • protein or peptide preparation is not pure; contaminant is more immunogenic than desired epitope; resulting antibody does not recognised the right target

See also

External links

Protocol Online (methods forum)