Increasing reliability by co-localization

Cell Atlas Co-localization HPA14 Immunocytochemistry Immunofluorescence Subcell Atlas Validation

Subcellular localization obtained using IF (left) and GFP (FP, right) for the endoplasmic protein FKBP7 (top) and the FHL2 protein localized to focal adhesion sites (bottom).

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

The Human Protein Atlas applies antibodies for a variety of applications to map protein expression in different tissues and also at the subcellular level. Within the subcellular protein atlas, immunofluorescence (IF) is used to uncover the localization of proteins to different organelles. To ensure an accurate localization of each and very protein, the antibodies have to be specific to their target protein. One way of strengthening the belief in an observed localization is to compare the obtained IF results with other imaging techniques not dependent on antibodies.

In a study published in Nature Methods, the localization of more than 500 proteins was compared from using in house produced antibodies (HPA antibodies) in fixed cells, and a green fluorescent protein (GFP) tagged variant of the target protein detected in live cells.

The localization obtained with IF and GFP tagging respectively were compared and scored to be identical (sharing one or several locations), similar (sharing at least one location but with additional locations observed in one of the two methods), or dissimilar (no locations shared between the methods). The analysis showed that 80% of the proteins yield the same subcellular distribution and that the majority of proteins localize to multiple organelles. In the dataset of 500 proteins, the locations of 250 previously unlocalized proteins could also be determined by the overlap between the two methods. In addition to the overall analysis of how similar the two methods are, systematic discrepancies between IF and GFPs could be identified.

The study concludes that both IF and GFP tagging are reliable techniques and based on this, the subcellular protein atlas is now using GFP tagged proteins to validate the IF results in a more systematic manner. The GFP data of >100 genes was recently published in the Human Protein Atlas version 14. Use the search field and select "gfp_validation:Supportive" to access the gene list.

Read the full article about increasing reliability by comparing IF and fluorescently tagged protein for subcellular protein localization.