A Western blot analysis is performed on a panel of human tissues and cell lines to evaluate antibody specificity. For antibodies with non-supportive result a revalidation using an over-expression lysate is performed.
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 supportive, uncertain or unreliable.
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.
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 (supportive, uncertain, or unreliable).
Band of predicted size in kDa (+/-20%) with additional bands present.
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, LY402729)
Target mass (kDa)
27.1, 21.2, 11.4
Immunocytochemistry is used to validate the antibody staining and for assessing and validating the protein expression pattern in selected human cell lines.
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; Supportive, Uncertain, or Unreliable, to represent the overall antibody staining in all analyzed cell lines.
Immunofluorescent staining of human cell line A-431 shows localization to endoplasmic reticulum.
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.