Antibody validation Practical guide to finding and validating suitable antibodies for research
1.A Identify the target antigen
- 1. Confirm the identity of the target antigen: Use GenecardThis integrated database of human genes provides concise genomic related information on all known and predicted human genes, Gene NamesIs a curated online repository of HGNC-approved gene nomenclature, gene families and associated resources including links to genomic, proteomic and phenotypic information or GenBankAn online repository of HGNC-approved gene nomenclature, gene families and associated resources including links to genomic, proteomic and phenotypic information to identify the approved nomenclature and any alternative names of the antigen. This is essential to enable effective searches for background information and identify any existing antibodies.
- 2. Confirm the protein sequence: The identification of the "canonical" protein sequence of your protein is another essential step that will allow you to obtain accurate information on your target. Use UniprotCentral repository of data on proteins and the leading universal protein resource to obtain the "canonical" protein sequence.
- 3. Identify the existence of variants/isoforms: Use Uniprot or AceViewAceView is the only database that defines the genes genome-wide by using the public experimental cDNA sequences from the same species. The analysis relies on the quality of the genome sequence and exploits sophisticated cDNA-to-genome co-alignment algorithms to provide a comprehensive and non-redundant representation of the GenBank, dbEST, GSS, Trace and RefSeq cDNA sequences to identify all known variants (caused by splicing, cleavage or post-translational modification, etc) of the protein and decide which are your specific targets for antibody detection in your experimental system. Multiple variants may cause additional bands in Western blots or altered patterns of tissue distribution and/or subcellular localisation that might otherwise be mistaken for non-specific reactivity.
- 4. Investigate the presence of related proteins to the target antigen: Proteins from the same family will share some degree of homology in their amino-acid sequence. It is also possible that your target protein may share some homology with unrelated proteins. Use BLAST searchesalgorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins to i) identify amino acid sequence conservation across species if you need to generate a monoclonal antibody against a common epitope distributed in multiple species. Regions with high sequence conservation across orthologous proteins are the most likely to bind antibodies raised in another species. ii) Identify related proteins, which share sequence identity with your target antigen, and which might also be able to bind your antibody.
- 5. Define ideal epitopes: Identify the region(s) of your target antigen to which you wish to raise an antibody of the desired specificity for your chosen molecule. This information is critical in antibody production but can also help you to predict the specificity of your antibody and its possible application. In fact, if an antibody has been raised using synthetic peptides it may not work well when a protein is in its native conformation. It could also be unable to detect post-translational modifications, since it will be recognising a linear epitope. Such antibodies may not be useful for immunoprecipitacion (IP) or immunohistochemistry (IHC) experiments, but will work well in Western Blotting (WB). The opposite could also be if the immunogen is a purified protein. In this case the antibody could work well for proteins in their native conformation, but not when denatured. In this case, WB cannot be an absolute standardization for antibody working in those assays where the antigen is in its native conformation.
Example step 1: How to define a chosen antigen for a monoclonal antibody to PIM2