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All assays through which the antibody has been validated. Assays&annotation provide a detailed description of the different assays. The pie-charts indicate degree of validation.
Immunohistochemistry is used for validating antibody reliability by assessing staining pattern in 44 normal tissues. Validation scores include Enhanced, Supported, Approved and Uncertain.
Validationi
Results of validation by standard or enhanced validation based on assessment of antibody performance in 44 normal tissues.
Standard validation results in scores Supported, Approved or Uncertain. An image representative of the antibody staining pattern is shown.
Enhanced validation results in the score Enhanced and includes two methods: Orthogonal validation and Independent antibody validation. For orthogonal validation, representative images of high and low expression are shown. For independent antibody validation, four images of each independent antibody are displayed.
Antigen retrieval is a method used to restore/retrieve the epitope (antibody bidning region) of the target protein, cross-linked, and thus masked, during tissue preserving fixative treatment of the tissues.
HIER pH6
Antibody dilution
1:1250
Literature conformityi
Conformance of the expression pattern with available gene/protein characterization data in scientific literature and data from bioinformatic predictions.
UniProt is used as the main source of gene/protein characterization data and when relevant, available publications and other sources of information are researched in depth. Extensive or sufficient gene/protein data requires that there is evidence of existence on a protein level and that a substantial quantity of published experimental data is available from literature and public databases. Limited protein/gene characterization data does not require evidence of existence on a protein level and refers to genes for which only bioinformatic predictions and scarce published experimental data is available.
Consistent with extensive gene/protein characterization data.
RNA consistencyi
Consistency between immunohistochemistry data and consensus RNA levels is divided into five different categories: i) High consistency, ii) Medium consistency, iii) Low consistency, iv) Very low consistency, and v) Cannot be evaluated.
High consistency between antibody staining and RNA expression data.
WESTERN BLOTi
A Western blot analysis is performed on a panel of human tissues and cell lines to evaluate antibody specificity. For antibodies with unreliable result a revalidation using an over-expression lysate is performed.
Validationi
Western Blot is used for quality control of the polyclonal antibodies generated in the project. After purification, the antibodies are used to detect bands in a setup of lysate and different tissues. The result is then scored Enhanced, Supported, Approved, or Uncertain.
Enhanced validation includes five different methods: Genetic validation, Recombinant expression validation, Independent antibody validation, Orthogonal validation and Capture MS validation.
Enhanced - Recombinant expressioni
This method is based on over-expression of the target protein in a cell line preferably not expressing the target protein. The staining of the antibody is evaluated by Western blot through analyses of samples from cell lysates with and without recombinant expression of the target protein. The results show no or weak band from the unmodified cell line lysate and a strong band in the cell line with recombinant expression.
Band of predicted size in kDa (+/-20%) with additional bands present.
Lane 1: Marker [kDa] 250, 130, 95, 72, 55, 36, 28, 17, 10 Lane 2: Negative control (vector only transfected HEK293T lysate) Lane 3: Over-expression Lysate (Co-expressed with a C-terminal myc-DDK tag (~3.1 kDa) in mammalian HEK293T cells, LY405465) Target mass (kDa): 38.9
Antibody dilution
1:250
PROTEIN ARRAY
Validationi
A protein array containing 384 different antigens including the antibody target is used to analyse antibody specificity. Depending on the array interaction profile the antibody is scored as Supported, Approved, or Uncertain.
Approved
Pass with quality comment low specificity (binding to 1-2 antigens >15% and <40%).
Antibody specificity analysis with protein arrays. Predicted and matching interactions are shown in green.
The Structure section provides predicted structures from the Alphafold protein structure database and includes
structures corresponding to uniprot entries mapped to our gene set with at least one splice variant having 100% identity to the structure sequence.
Displaying protein features on the AlphaFold structures
Individual splice variants can be selected in the top part of the Protein Browser (see below) and both for transcripts matching the whole structure and those corresponding only to a part the full-length AlphaFold structure is shown.
Different transcript-related features such as transmembrane regions, InterPro domains and antigen sequences for antibodies can be displayed in the structure by clicking on the respective features in the Protein Browser and then also the part of the structure corresponding to the selected transcript will be shown in lightblue. Clinical and population amino acid variants can be highlighted by using the sliders to the right of the structure, which can also be used to colour the entire structure by residue index or make the structure autorotate.The structures are displayed using the NGL Viewer and can also be zoomed-in and rotated manually.
The Protein Browser
The protein browser displays the antigen location on the target protein(s) and the features of the target protein. The tabs at the top of the protein view section can be used to switch between the different splice variants to which an antigen has been mapped.
At the top of the view, the position of the antigen (identified by the corresponding HPA identifier) is shown as a green bar. A yellow triangle on the bar indicates a <100% sequence identity to the protein target.
Below the antigens, the maximum percent sequence identity of the protein to all other proteins from other human genes is displayed, using a sliding window of 10 aa residues (HsID 10) or 50 aa residues (HsID 50). The region with the lowest possible identity is always selected for antigen design, with a maximum identity of 60% allowed for designing a single-target antigen (read more).
The curve in blue displays the predicted antigenicity i.e. the tendency for different regions of the protein to generate an immune response, with peak regions being predicted to be more antigenic.The curve shows average values based on a sliding window approach using an in-house propensity scale. (read more).
If a signal peptide is predicted by a majority of the signal peptide predictors SPOCTOPUS, SignalP 4.0, and Phobius (turquoise) and/or transmembrane regions (orange) are predicted by MDM, these are displayed.
Low complexity regions are shown in yellow and InterPro regions in green. Common (purple) and unique (grey) regions between different splice variants of the gene are also displayed (read more), and at the bottom of the protein view is the protein scale.
The Human Protein Atlas project is funded
