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

Abstract

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.

Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

“…Although the other 12 genes showed various expression patterns, including ubiquitous expression, cytoplasmic localization, or nuclear localization, it is possible that they also localize to mitochondria, as many mitochondrial proteins do not exclusively reside in mitochondria (14). In addition, artificially tagging GFP to a protein may disrupt its structure and thus affect its endogenous localization (15). Alternatively, these 12 genes may represent false positives from the APEX approach.…”

Proteome-wide Epitope Mapping of Antibodies Using Ultra-dense Peptide Arrays

“…Antibodies are used in proteomics both as imaging reagents for the analysis of tissue specificity (1) and subcellular localization (2) and as capturing agents for targeted proteomics (3), in particular for the enrichment of peptides for immunoaffinity methods such as Stable Isotope Standards and Capture by Anti-peptide Antibodies (4). In fact, the Human Proteome Project (5) has announced that one of the three pillars of the project will be antibody-based, with one of the aims being to generate antibodies to at least one representative protein from all protein-coding genes.…”

A Fluorescent Live Imaging Screening Assay Based on Translocation Criteria Identifies Novel Cytoplasmic Proteins Implicated in G Protein-coupled Receptor Signaling Pathways

“…The use of fluorescently tagged overexpressed proteins to probe intracellular localization was validated by protein correlation profiling although less correlation was observed for cytosolic proteins (9). More recently, the Ͻ Ͻprotein AtlasϾ Ͼ project has found a good correlation between endogenous proteins localization using immunofluorescence techniques compared with intracellular localization of the same proteins as determined in the GFP-cDNA localization project (10). Therefore, the cDNA library of this GFP-cDNA localization project was selected for our live-imaging screen: from this library, we screened the proteins that had been identified residing in the cytoplasm or cytoplasm and nucleus.…”