Antibodies that fulfil the criteria are labelled "Enhanced". The enhanced validation principles are adapted for validation in Western blot, immunocytochemistry and immunohistochemistry applications. For further validation we refer to quality controls provided by the respective company.ĭetailed descriptions of the strategies used for standard antibody validation in the different assays are available further down on this page.Īntibodies used for Western blot, immunocytochemistry and immunohistochemistry in the Human Protein Atlas undergo enhanced antibody validation based on the five "pillars" described by the International Working Group for Antibody Validation (IWGAV), presented in "A proposal for validation of antibodies" ( Uhlen M et al. For each commercially available antibody, a link to the antibody provider is given on the "Antibody validation" page. These antibodies have also been tested on Western blot in a standardized setup. High resolution confocal microscopy images of human cell lines stained by indirect immunofluorescence are annotated for subcellular localizations by trained cell biologists, and the subcellular localization patterns are compared with the immunohistochemical staining and available experimental protein characterization data.įor antibodies supplied through commercial or other academic sources (CAB antibodies), immunocytochemistry and immunohistochemistry have been performed and validated in a similar manner as for HPA antibodies.Immunohistochemical staining of normal and cancer tissue is examined and annotated by specially educated personnel, and the staining patterns are compared with available gene/RNA/protein characterization data.Antibodies with an uncertain standard Western blot are reanalyzed using an over-expression lysate as a positive control. Total protein lysates from a limited number of tissues (liver and tonsil), cell lines (RT4 and U-251 MG), and human plasma are used to evaluate the antibody target binding in a Western blot setting. Antibody specificity is analyzed using Western blot in a standardized setup.To control for cross-reactivity, affinity purified antibodies are tested for sensitivity and specificity on protein arrays consisting of glass slides with spotted PrEST fragments.Size of the resulting recombinant protein (including the specific PrEST) is analyzed using mass spectrometry to assure that the correct antigen has been produced and purified.Plasmid inserts are sequenced to assure that the correct PrEST sequence is cloned.Multitarget PrESTs are PrESTs that have more than 80% identity to proteins from more than one gene, and are expected to generate antibodies with multiple targets. The antigen (protein epitope signature tag (PrEST)) for a protein is selected as a stretch of 20-150 amino acids with as low identity as possible to proteins from all other putative protein-coding genes, and not including signal peptides or transmembrane regions.Quality assurance steps for antibodies generated within the Human Protein Atlas project: Feedback from the research community is appreciated and needed for continuous curation of data. All antibodies that provide a reasonable pattern of immunoreactivity are added to the Human Protein Atlas portal. Steps 5-7 provide the basis for evaluating and scoring the antibody reliability. All antibodies produced internally within the Human Protein Atlas project (HPA antibodies) must pass steps 1-4 in the list below in order to be used for immunohistochemistry and immunocytochemistry/IF.
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