Figure 1.

Schematic overview of recombinant p-S65-Ub antibody clone generation, selection, and validation. The illustration summarizes the entire process of recombinant, rabbit monoclonal antibody production starting from testing of the initial polyclonal bleeds to the isolation and screening of B cells, and the subsequent cloning of variable heavy and light chain antibody regions into an IgG vector frame. Recombinant antibodies were then sequenced, expressed in HEK293 cells and supernatants were screened by western blot and ICC. The five most promising antibody clones were affinity purified and re-validated by a series of biochemical and imaging analyses using recombinant proteins as well as cells and brain tissues from mouse and human origin. PBMC: peripheral blood mononuclear cell; ELISA: enzyme-linked immunosorbent assay; ICC: immunocytochemistry; IHC: immunohistochemistry. Created with BioRender.com.