Immunofluorescence and fluorescent protein-tagging are complementary techniques with high correlation for subcellular investigation of the human proteome in mammalian cells
(English)Article in journal (Other academic) Submitted
Imaging techniques such as immunofluorescence (IF) and expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Due to potential steric hindrance with FPs and fixation artifacts obtained during IF, the reliability of these two fundamental methods is often discussed. Here we report a systematic study of over 500 human proteins where the localizations obtained in live versus fixed cells using FPs and IF respectively have been compared. The results indicate that 80% of the analyzed proteins yield the same subcellular distribution, which is as high as that seen for FP tagging at either the N- and C-terminal. The localizations of 250 proteins, with no previous experimental data, were determined by the overlap of the two methods as applied here. The fraction of proteins located to multiple organelles is approximately 60% for both methods, indicating a complex subcellular protein organization. The result shows that IF and FP tagging are reliable techniques, both needed for a complete investigation of the subcellular human proteome.
IdentifiersURN: urn:nbn:se:kth:diva-103628OAI: oai:DiVA.org:kth-103628DiVA: diva2:561089
QS 20122012-10-172012-10-172012-10-17Bibliographically approved