Results of validation by standard or enhanced validation.
Standard validation is based on concordance with available experimental gene/protein characterization data in the UniProtKB/Swiss-Prot database. Standard validation results in scores Supported, Approved or Uncertain.
Enhanced validation is performed using either siRNA knockdown, tagged GFP cell lines or independent antibodies. For the siRNA validation the decrease in antibody-based staining intensity upon target protein downregulation is evaluated. For the GFP validation the signal overlap between the antibody staining and the GFP-tagged protein is evaluated. For the independent antibodies validation the evaluation is based on comparison of the staining of two (or more) independent antibodies directed towards independent epitopes on the protein.
For all cases except the siRNA validation, an image representative of the antibody staining pattern is shown. For the siRNA validation, a box plot of the results is shown.
The subcellular location is supported by literature.
Immunofluorescent staining of human cell line U2OS shows localization to nucleoplasm.
Human assay: A-549 fixed with PFA, dilution: 1:26 Human assay: CACO-2 fixed with PFA, dilution: 1:26 Human assay: U2OS fixed with PFA, dilution: 1:26 Mouse assay: NIH 3T3 fixed with PFA, dilution: 1:26
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.
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.
This method is based on comparison between the molecular weight of the stained band visualized by the antibody against the protein size obtained by a capture MS method in which multiple gel slices are cut out from the electrophoretic separation of cell lysates of RT4 and U-251 and analysed separately by proteomics. The band detected by the antibody should be equivalent to the same of the intended target protein and its peptide(s).
Antibody band pattern is confirmed by capture-MS.
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.
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.