0.5 μL of siRNA stock solution and 1 μL of INTERFERin were mixed in 50 μL of Opti-MEM, and incubated for 10 min. Transfection mix was added to the cells, together with 150 μL of fresh complete growth media for a final siRNA working concentration of 10 nM.

Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in immunofluorescence applications using endogenous expression of tagged proteins. This is an important step towards a reproducible approach for context and application specific antibody validation and improved reliability of antibody-based experiments and research data.