Enhanced validation of antibodies for research applications

Abstract

There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.

Fig. 1

Advantages and disadvantages of the five strategies used for enhanced Western blot antibody validation. Throughput is a relative estimate of the possibility to use the method for high-throughput settings and specificity is the estimate of how reliable the validation results are to determine the specificity of the antibody. Low: +, Medium: ++, High: +++

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. 2–3 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

Fig. 3

Validation of antibodies using capture MS. a Principle for validation of antibodies based on separation by SDS-PAGE and comparing the protein migration profile determined by an antibody (Western blot) and peptide identification performed by proteomics (capture MS). b Examples of orthogonal validation of two antibodies by capture MS in two different cell lines (RT4, U-251). The black arrow indicates theoretical molecular weight, and the blue bars represent number of peptides identified in each gel slice. c Summary of capture MS validation of antibodies (n = 2,888) showing if they were validated using other antibody validation pillars. d Distribution of theoretical molecular weights for the largest transcript of all (n = 19,628) human protein-coding genes divided into intracellular (blue) and secreted and membrane bound proteins (purple). Molecular weights are represented by the log₂ value together with a molecular weight ladder from a typical Western blot assay used within the Human Protein Atlas program

Fig. 4

Summary of the five-pillar strategy used for antibody validation. a Summary of the five-pillar strategy employed within the Human Protein Atlas with the number of enhanced validated antibodies together with gene-coverage in each respective category. b Heat map showing 267 antibodies with enhanced validation (blue) in more than three of the five pillars. The two other validation states include not done (black) and uncertain (gray)