We use cookies to enhance the usability of our website. If you continue, we'll assume that you are happy to receive all cookies. More information. Don't show this again.
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.
Immunocytochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in selected human cell lines.
Validationi
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.
Enhanced - Independent antibodiesi
This method is based on comparing the staining pattern using two independent antibodies with no overlapping epitopes. A similar staining pattern (main and additional locations) results in an enhanced antibody validation.
Antibody staining overlaps with antibody HPA056900.
Immunofluorescent staining of human cell line Hep-G2 shows localization to nucleoplasm. Protein location across all samples similar between independent antibodies
Enhanced - Independent antibodiesi
This method is based on comparing the staining pattern using two independent antibodies with no overlapping epitopes. A similar staining pattern (main and additional locations) results in an enhanced antibody validation.
Antibody staining overlaps with antibody HPA040062.
Immunofluorescent staining of human cell line RT-4 shows localization to nucleoplasm. Protein location across all samples similar between independent antibodies
Antibody dilution
Human assay: Hep-G2 fixed with PFA, dilution: 1:6 Human assay: RT-4 fixed with PFA, dilution: 1:6 Human assay: U2OS fixed with PFA, dilution: 1:6
Human assay: Hep-G2 fixed with PFA, dilution: 1:60 Human assay: RT-4 fixed with PFA, dilution: 1:60 Human assay: U2OS fixed with PFA, dilution: 1:60
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.
Uncertaini
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.
No bands detected. Analysis performed using a standard panel of samples.
250
130
95
72
55
36
28
17
10
Supportedi
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.
Single band corresponding to the predicted size in kDa (+/-20%). Analysis performed using a standard panel of samples.
250
130
95
72
55
36
28
17
10
Antibody dilution
1:250
1:200
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.
Supported
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.
Supported
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 in-house generated structures, predicted using the Alphafold source code, for the majority of the proteins and their related isoforms.
Displaying protein features on the AlphaFold structures
Individual splice variants can be selected in the top part of the Protein Browser (see below) and 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.
Clinical and population-based amino acid variants based on data from the Ensembl variation database and AlphaMissense (AM) predictions can be highlighted using the sliders to the right of the structure. These 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 ProteinBrowser displays the antigen location on the target protein(s) and the features of the target protein. Transcript names and schematic transcript structures including exons, introns and UTRs for the different isoforms are shown on top, and can be used to switch between the structures for the different splice variants.
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).
Signal peptides (turquoise) and membrane regions (orange) based on predictions using the
majority decision methods
MDM and MDSEC are also 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
