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Immunocytochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in selected human cell lines.
Immunohistochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in normal human tissues. For validation of antibody staining the immunohistochemical staining patterns obtained using tissue microarrays (TMA) containing 44 different normal tissue types (IHC tissue) , as well as, cell microarrays (CMA) containing 44 different widely used and well characterized human cell lines (IHC cells) , are compared to staining patterns from independent antibodies or to RNA expression patterns. For validation of protein expression patterns in normal human tissues the TMA staining patterns are evaluated together with RNA-seq data from internal and external sources and available protein/gene characterization data.
A Western blot analysis is performed on a panel of human tissues and cell lines to evaluate antibody specificity. For antibodies with uncertain result a revalidation using an over-expression lysate is performed.
Immunocytochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in selected human cell lines.
Tagged proteins: the antibody specificity has been validated by co-staining of a cell line expressing the target protein tagged with EGFP at near-endogenous levels.
Antibody staining overlaps with GFP tagged protein
Figure description
Immunofluorescent staining of transgenic HeLa cells show antibody staining in nucleus but excluded from the nucleoli and GFP expression in nucleus but excluded from the nucleoli.
Antibody dilution
1:114
Standard validationi
For each antibody, the observed staining in the different cell lines is assigned a validation score based on concordance with available experimental gene/protein characterization data in the UniProtKB/Swiss-Prot database. The validation scores for up to three cell lines are merged into one of the main categories; Supported, Approved, or Uncertain to represent the overall antibody staining in all analyzed cell lines.
Immunofluorescent staining of human cell line U-2 OS shows no positivity.
Antibody dilution
1:114
Literature conformityi
The immunostaining patterns is compared for consistency with available experimental gene/protein characterization data in the UniProtKB/Swiss-Prot database and other experimental evidence for location described in scientific literature.
The subcellular location is partly supported by literature or no literature is available.
IMMUNOHISTOCHEMISTRYi
Immunohistochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in normal human tissues. For validation of antibody staining the immunohistochemical staining patterns obtained using tissue microarrays (TMA) containing 44 different normal tissue types (IHC tissue) , as well as, cell microarrays (CMA) containing 44 different widely used and well characterized human cell lines (IHC cells) , are compared to staining patterns from independent antibodies or to RNA expression patterns. For validation of protein expression patterns in normal human tissues the TMA staining patterns are evaluated together with RNA-seq data from internal and external sources and available protein/gene characterization data.
For each antibody the observed staining pattern is assigned a validation score based on the conformance with UniProt gene/protein characterization data, as well as, the consistency with RNA expression. The validation score levels are supported, approved, and uncertain.
Immunohistochemical staining of human kidney shows strong nuclear positivity in tubules and glomeruli.
Expression
RNA: detected in 37 tissues Protein: detected in 75 cell types
Retrieval
HIER pH6
Antibody dilution
1:25
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 internally generated RNA-seq data is divided into five different categoreies: i) Consistent with RNA expression data, ii) Mainly consistent with RNA expression data, iii) Mainly not consistent with RNA expression data, iv) Not consistent with RNA expression data, and v) No internal RNA expression data available for correlation.
Mainly consistent with 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.
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 Supported, Approved, or Uncertain.
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.
