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Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells


Imaging techniques such as immunofluorescence (IF) and the expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Here we report a systematic analysis of >500 human proteins comparing the localizations obtained in live versus fixed cells using FPs and IF, respectively. We identify systematic discrepancies between IF and FPs as well as between FP tagging at the N and C termini. The analysis shows that for 80% of the proteins, IF and FPs yield the same subcellular distribution, and the locations of 250 previously unlocalized proteins were determined by the overlap between the two methods. Approximately 60% of proteins localize to multiple organelles for both methods, indicating a complex subcellular protein organization. These results show that both IF and FP tagging are reliable techniques and demonstrate the usefulness of an integrative approach for a complete investigation of the subcellular human proteome.

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Figure 1: Overlap between IF and FP localization data.
Figure 2: Differences between IF and FP localization data.
Figure 3: Validation of antibody-based localization data.
Figure 4: Differences between N- and C-terminal FP fusions.
Figure 5: Feature-based image analysis of IF and FP patterns.
Figure 6: Systematic localization of uncharacterized proteins.

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The authors wish to acknowledge the entire staff of the Human Protein Atlas project, S. Wiemann and his lab (German Cancer Research Center, DKFZ) for various GFP-ORF constructs and S. Simpson for careful proofreading. The IF work within the frame of the Human Protein Atlas project was supported by grants from the Knut and Alice Wallenberg Foundation, EU Seventh Framework Programme (GA HEALTH-F4-2008-201648/PROSPECTS) and strategic grant Science for Life Laboratory. The J.C.S. lab is supported by a Principal Investigator (PI) grant (09/IN.1/B2604) from Science Foundation Ireland (SFI).

Author information

Authors and Affiliations



C.S. and E.L. provided the IF data. J.C.S. and R.P. provided the FP data. C.S. and E.R. performed the comparisons between the data sets. E.R. performed the automated image analysis. C.S. and V.R.S. performed control experiments. M.U. and R.F.M. provided intellectual input. E.L. designed and led the study. E.L., J.C.S. and C.S. wrote the manuscript.

Corresponding author

Correspondence to Emma Lundberg.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Notes 1 and 2 (PDF 12928 kb)

Supplementary Table 1

All N- and C-terminal FP fusions and corresponding localizations (XLSX 77 kb)

Supplementary Table 2

All antibodies used for IF and the corresponding localizations (XLS 109 kb)

Supplementary Table 3

All proteins with dissimilar results between IF and FP with the corresponding annotation and, if available, information on subcellular localization from UniProtKB (XLSX 34 kb)

Supplementary Table 4

All proteins with overlapping subcellular localizations for IF and FP and information on subcellular localization from UniProtKB (XLS 102 kb)

Supplementary Data 1

Top 50 features extracted from the IF and FP-tagged confocal microscopy images and used for the principal-component learning in the training data set and for the projection of the data in the validation data set (TXT 4 kb)

Supplementary Data 2

Labels of the dendrograms in Figure 5. First column is dendrogram in a, the second column is dendrogram in b and the third column is dendrogram in c. (TXT 12 kb)

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Stadler, C., Rexhepaj, E., Singan, V. et al. Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells. Nat Methods 10, 315–323 (2013).

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