Generating antibodies

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== Peptide selection ==
== Peptide selection ==
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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 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. Still, peptide selection is trial and error.
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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. Still, peptide selection is trial and error.
== Boosting the immune reaction ==
== Boosting the immune reaction ==

Revision as of 10:13, 23 April 2008

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

Contents

Early decisions

Type of antibody

polyclonal

  • faster to generate
  • less specific
  • cheaper than monoclonals

monoclonal

  • 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

  • rabbit
  • goat
  • mouse,..

Depends on choice of antibody (monoclonal requires mouse) and antibodies to be used in conjunction with antibody to be generated.

Type of antigen to use

  • full-length protein (natural selection of epitope, epitope location initially unknown, may not be availalbe, epitope may not be specific to protein)
  • peptide (cheaper, can typically be synthesised without problems, known epitope, possibly low antigenicity)

Peptide selection

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. Still, peptide selection is trial and error.

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. [1] See also adjuvant in the wikipedia.

See also

External links

Protocol Online (methods forum)

Wikipedia

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