Systematic antibody validation using siRNA

Cell Atlas Gene editing Immunocytochemistry Immunofluorescence siRNA Subcell Atlas

Fig 1a. The protein HNRNPUL1 is localized to the nucleus by staining with antibody HPA046290 (green)
Fig 1b. The antibody signal is significantly decreased after siRNA mediated knock-down of HNRNPUL1
Fig 2a. The protein AXTN2 is localized to the cytoplasm by staining with antibody HPA018295 (green)
Fig 2b. The antibody signal is significantly decreased after siRNA mediated knock-down of ATXTN2

Systematic antibody validation with siRNA for the Human Protein Atlas

Antibodies are among the most frequently used tools for basic research and clinical assays. For antibodies used in therapy or diagnostics, there are well-defined and strict guidelines that must be complied with before approval for clinical assays. For research antibodies, such guidelines have not yet been developed, despite the importance of demonstrating that they are specific, selective, and yield reproducible results in the immunoassay for which they are to be used.

Recent advancements of genetic tools that transiently or permanently regulate gene expression offer new possibilities for antibody validation. Within the Human Protein Atlas, antibodies are systematically validated using siRNA knockdown assays. On the Atlas, siRNA mediated decreasions of antibody signal are displayed for a quantitative immunofluorescence protocol, as well as for cell lysate western blots. For both assays, the starting point is a reverse solid phase transfection protocol employed to produce siRNA coated multi well plates suitable for high throughput downstream immunoassays.

For validated antibodies, the results of the quantitative immunofluorescence assay are accessed under the protein's page in the SUBCELL Atlas by clicking the "SUBCELL siRNA" toggle to the left. Alongside 10x and 40x images from siRNA- and control wells, results from the quantitative image analysis are visualized in box plots. When browsing the images, make sure to activate intensity color look-up table to better visualize the changes in fluorescent signal caused by the siRNA transfection.

Results from western blot assays are presented on the protein's "ANTIBODY/ANTIGEN" page, accessed through the menu on the left hand side.

Have a look at these examples of successfully siRNA-validated antibodies for an illustration of the methods used:

  • TOP2B
  • VARS
  • ATXN2
  • UBTF

    190 genes have been silenced and analyzed using western blot.

    233 genes have been silenced and analyzed using immunocytochemistry.

    Read the full story about the Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscop by Stadler et al.

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