Technical Aids

Human Protein Atlas (HPA) Project Workflow

The HPA is currently exploring the human proteome through antibody-based proteomics, which combines the high-throughput generation of monospecific antibodies with protein profiling in standard sets of tissue and cell microarrays. Each antibody produced begins with the selection of a protein epitope signature tag (PrEST). The PrEST fragment is typically 50-150 amino acids in length and is as dissimilar as possible to other proteins. The recombinant PrEST protein is utilized as the antigen in generating affinity purified monospecific antibodies.

The antibodies are then thoroughly tested through a series of quality assurance steps. Plasmid inserts are sequenced to ensure that the correct PrEST sequence is cloned. The size of the resulting recombinant protein is analyzed using mass spectrometry to ensure that the correct antigen has been produced and purified. To ensure antigen specificity, the antibodies are tested on protein arrays spotted with PrEST fragments. All 700 IHC staining images in normal and disease tissues, cell lines and cell types are annotated and curated by certified pathologists or by specially educated research scientists. In addition, the antibodies are tested by Western blot analysis and immunofluorescent microscopy. The antibodies are then assigned a validation score that assesses all validation tests as well as bioinformatic predictions and literature data. All of the images and data are available publicly on the Human Protein Atlas website www.proteinatlas.org.

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