Generating antibodies

Drawing of an antibody - heavy chain red, light chain 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 (faster to generate, less specific)
• monoclonal (slower to generate, more specific, after initial setup no animals required)

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)

See also

• Protein purification tags
• Western blot

External links

• Guidelines for polyclonal antibody production by Animal Care and Use Committee, UC Berkeley
• Introduction to monoclonal antibody generation by Mark Soloski, Johns Hopkins
• FAQ on antibody generation by Alta Bioscience, UK
• 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

Wikipedia

• Antibody, esp. research applications
• Polyclonal antibodies, Monoclonal antibodies