Fig. 2 | Nature Communications

Fig. 2

From: Enhanced validation of antibodies for research applications

Fig. 2

Orthogonal validation of antibodies using proteomics. a Principle for the Western blot validation strategy based on correlating protein expression levels determined across a panel of cell lines using either proteomics or transcriptomics. b Example of orthogonal validation of Western blot bands (WB, relative intensity) by proteomics (Parallel Reaction Monitoring, PRM) reported as ratio to standard or transcriptomics reported as Transcript Per Million (TPM). Error bars represent 1 s.d. across three technical replicates. The black arrow indicates the theoretical molecular weight of the protein and blue arrows indicate the band subjected for the intensity-based relative quantification to determine the antibody staining profile. More examples including loading controls are presented in Supplementary Fig. 23 for PRM and TMT, respectively. c Mirror plot summarizing the Pearson’s r for 53 antibodies evaluated either by TMT (dark blue) or by PRM (light blue) including transcriptomics-based validation based on RNA expression (purple). d Analysis of the Pearson’s r between Western blot band intensities and RNA expression levels as a consequence of the fold-change between the highest and lowest value across the cell lines. The gray area represents fold-change in RNA levels less than fivefold. Antibodies in the green area (Pearson’s r > 0.5) are considered validated while antibodies in the red area (Pearson’s r < 0.5) are considered not validated