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Antibody validation Practical guide to finding and validating suitable antibodies for research

3.B Its suitability for use in your intended applications

  • 1. Some reagents work in a wide number of techniques whereas others are very limited in their application: Ideally choose an antibody that has been validated, if possible in the same species and tissue type, for the technique you want to use. If this information is unavailable then further experimental testing is strongly advised.
  • 2. Problems of cross-reactivity: There are some antibodies that may be specific in one technique only.
  • 3. Alternative choices: Consider whether it is better to choose an antibody that is more likely to be specific for your target antigen rather than one that the supplier recommends for a particular technique.
  • 4. Evidence for suitability in any related applications: An antibody might have been tested using only a small number of techniques but this does not mean that it does not work in other applications. For example, an antibody recommended for immunofluorescence may also work in immunohistochemistry on frozen tissues. Alternatively an antibody that recognises a formalin resistant epitope by immunohistochemistry may also work in chromatin immunoprecipitation, which also requires the recognition of a formalin resistant epitope. Many antibodies are primarily validated for Western blotting in which a denatured antigen is usually recognised and its molecular weight, isoforms and subcellular location can be assessed. Antibodies that recognise only the natural conformation of the protein may not, therefore, work in this technique.