Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

A resource for improved antibody validation (#570)

Andreas Hober 1 , Kalle von Feilitzen 1 , Anna Sjöberg 2 , Tove Boström 2 , Marie Utterbäck 2 , Björn Forsström 1 2 , Fredrik Edfors 1 , Mathias Uhlen 1
  1. Department of Protein Science, Science for Life Laboratory, KTH - Royal Institute of Technology, Stockholm, Sweden
  2. Atlas Antibodies, Stockholm, Sweden

Antibodies are of great importance within many different areas of proteomics research as well as within clinical diagnostics. Using affinity reagents as the mean of detection puts a high demand on the reagents when it comes to selectivity and specificity. This has been illuminated by the International Working Group for Antibody Validation (IWGAV), which suggest that an antibody must be evaluated in an application-specific manner. When an antibody is to be used within a Western blot assay, the initial measure for evaluating its specificity is the theoretical molecular weight of its target protein, or rather the migration of a set of reference proteins during gel electrophoresis. It is well known that the migration of proteins during gel electrophoresis can differ from the standards used within the ladder. A dataset of migration patterns for more than 8,000 proteins across nine cell lines and tissues has been established by characterizing the respective proteins migration pattern by mass spectrometry. The dataset has been benchmarked against more than 200 siRNA validated antibodies and more than 6,000 antibodies from the Human Protein Atlas have been evaluated using the data. This strategy provides a more accurate way of evaluating antibodies based on the actual migration of their target protein rather than a theoretical molecular weight and also highlights the importance of validating antibodies in an application-specific manner.