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.
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 localization to vesicles.
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 not consistent with literature.
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.
Synaptic staining in many brain regions. Strongest immunoreactivity in hippocampus and the granular layer of the cerebellum.
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.
Single band corresponding to the predicted size in kDa (+/-20%).
Analysis performed using a standard panel of samples. No bands detected.
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, LY422121)
Lane 1: Marker [kDa] 250, 130, 95, 72, 55, 36, 28, 17, 10 Lane 2: RT4 Lane 3: U-251 MG Lane 4: Human Plasma Lane 5: Liver Lane 6: Tonsil
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.