Immunocytochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in selected human cell lines.
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.
Reliability scores for antibodies used in immunocytochemistry are set by comparing the staining pattern in cell lines with external experimental evidence for protein localization. The scores are termed Supported, Approved and Uncertain.
The subcellular location is partly supported by literature or no literature is available.
Immunofluorescent staining of human cell line U-251MG shows localization to mitochondria.
Human assay: Rh30 fixed with PFA, dilution: 1:20 Human assay: U-251MG fixed with PFA, dilution: 1:20 Human assay: U2OS fixed with PFA, dilution: 1:20
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.
The staining of an antibody is evaluated by Western Blot through analysis of samples from different cell lysates. A supportive score is given if band(s) of predicted size in kDa (+/-20%) is detected.
Band of predicted size in kDa (+/-20%) with additional bands present. Analysis performed using a standard panel of samples.
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.
Pass with single peak corresponding to interaction only with its own antigen.
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.