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.
Type of antibody
- faster to generate
- less specific
- cheaper than monoclonals
- 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
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)
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. For example, several copies of the peptide linked as branches to a carrier peptide chain
- Adjuvants are mixtures co-injected with the antigen to stimulated the immune response. Complete Freund's adjuvant (CFA), typically made from inactivated mycobacteria, is a common choice. However, newer adjuvants like Ribi's adjuvent are safer for both scientist and animal.  See also adjuvant in the wikipedia.
- Guidelines for polyclonal antibody production by Animal Care and Use Committee, UC Berkeley
- FAQ on antibody generation by Alta Bioscience, UK (2 pages of useful technical tips and a little advertisement)
- Backgrounder on history, generation, and application of monoclonal antibodies by Mark Soloski, Johns Hopkins
- Hybridoma and in vitro generation of antibodies, Antibody Facility, UNC Chapel Hill
- Monoclonal Antibodies: A Practical Approach by Philip Shepherd, Christopher Dean on Google Book Search
- Protocol for raising polyclonal antibodies in rabbits, Chew lab, Uni of Singapore
Protocol Online (methods forum)
- Protocol-online.org archive, keyword antibody and discussions indexed under antibody
- Discussion on affinity purification
- Discussion on antigen amount