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A high-throughput pipeline for validation of antibodies


Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).

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Fig. 1: A high-throughput pipeline for antibody validation.
Fig. 2: PAGE-MAP offers high precision for the comparison of antibody reactivity profiles.
Fig. 3: PAGE-MAP facilitates direct assessment of antibody specificity by PAGE–IP-MS.

Data availability

Supplementary Tables 29 contain all data, and an overview is presented in Supplementary Table 1. Raw MS data were uploaded to PRIDE under accessions PXD005945 and PXD010510. Line charts with PAGE-MAP data for antibodies that passed validation and MS data for the intended targets were uploaded to Identifier data for all antibodies tested in the study can be obtained on reasonable request from the corresponding author.


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The authors thank J. Olweus, K. Tasken, K.-J. Malmberg and E. Marcon for critical reading. HeLa cells were a kind gift from M.S. Rødland (Oslo University Hospital, Oslo, Norway). This work was funded by grants from the KG Jebsen Foundation to the KG Jebsen Centre for Immunotherapy of Cancer and the KG Jebsen Inflammation Research Centre, Helse-Sør-Øst, Novo-Nordisk Foundation, and the Norwegian Research Council.

Author information

Authors and Affiliations



K.S., A.M., M.I. and F.T. conducted experiments (PAGE-MAP), analyzed data and prepared figures. S.K. and M.T carried out DigiWest experiments and data analysis. T.K., J.S. and W.Z. analyzed data. T.A.N., M.E.S. and G.A.d.S. performed mass spectrometry experiments. L.H. performed statistical analysis. F.L.-J. designed the experiments, analyzed data, contributed to figure preparation and wrote the manuscript.

Corresponding author

Correspondence to Fridtjof Lund-Johansen.

Ethics declarations

Competing interests

M.T. is associated with NMI TT GmbH, a company that sells DigiWest analysis as a service. W.Z. is the founder of Seekquence, a company that sells information about research reagents including antibodies.

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Integrated supplementary information

Supplementary Figure 1 Antibody sensitivity is linked to reproducibility.

The box plots show signal-to-noise ratios of antibodies that clustered according to specificity in one experiment only or in both experiments. Whiskers indicate lower and upper quartiles. The Venn diagram shows the number of antibodies in each category. Source data: Supplementary Table 2.

Supplementary Figure 2 PAGE-MAP yields reproducible data for antibody sensitivity.

The scatter plot to the left shows signal-to-noise ratios measured for 3,672 antibodies in two biological replicates. The plot to the right shows ratios measured for 1,712 antibodies in two technical replicates. R values indicate Pearson correlations. Source data: Supplementary Table 5.

Supplementary Figure 3 Shotgun MS signals in the cell type used as the source for IP and signal intensity in single-plex PAGE-IP-FCM are predictive for target identification by PAGE-IP-MS.

(a) A subset of beads from each single-plex IP were labeled with streptavidin–phycoerythrin and analyzed by FCM. The box plots show streptavidin fluorescence intensity of beads in IPs where the intended target was identified (blue) or not (red). (b) Shotgun MS signals measured in cell types used as sources for IPs. Blue and red shading indicates results for antibody targets that were detected and not detected by PAGE-IP-MS, respectively. Numbers in parentheses indicate the number of antibodies in each category. Whiskers indicate lower and upper quartiles. Source data: Supplementary Table 7.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Data

Reporting Summary

Supplementary Table 1

Overview of all supplementary material

Supplementary Table 2

Antibody-array and MS data

Supplementary Table 3

MS source data, MS metadata and assessment of P values for correlations

Supplementary Table 4

PAGE-MAP technical replicates

Supplementary Table 5

Reproducibility of signal-to-noise ratios in PAGE-MAP

Supplementary Table 6

Shotgun MS reproducibility

Supplementary Table 7

PAGE–IP-MS results

Supplementary Table 8

PAGE-MAP versus DigiWest and WB

Supplementary Table 9

List of antibodies that passed validation in PAGE-MAP

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Sikorski, K., Mehta, A., Inngjerdingen, M. et al. A high-throughput pipeline for validation of antibodies. Nat Methods 15, 909–912 (2018).

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