Fluorescent Proteins, Anna Jost (NIC)

Cranfill, P. J., Sell, B. R., Baird, M. A., Allen, J. R., Lavagnino, Z., de Gruiter, H. M., … Piston, D. W. (2016). Quantitative assessment of fluorescent proteins. Nature Methodshttp://doi.org/10.1038/nmeth.3891

  •            Measures standard properties for a comprehensive selection of FPs under comparable conditions. A good starting point for generating a list of FPs to test in your system.

        Heppert, J. K., Dickinson, D. J., Pani, A. M., Higgins, C. D., Steward, A., Ahringer, J., … Goldstein, B. (2016). Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system. Molecular Biology of the Cell27(22), 3385–3394. http://doi.org/10.1091/mbc.E16-01-0063

  •            Compares FP performance in C elegans embryos and finds that the FPs reported to have the best properties in vitro do not always perform better in vivo.

        Costantini, L. M., Fossati, M., Francolini, M., & Snapp, E. L. (2012). Assessing the Tendency of Fluorescent Proteins to Oligomerize Under Physiologic Conditions. Traffic13(5), 643–649.

  •     Initial publication of the OSER assay, which uses fusions of FPs to an ER protein called CytERM to test monomericity of FPs in vivo

        Landgraf, D., Okumus, B., Chien, P., Baker, T. a, & Paulsson, J. (2012). Segregation of molecules at cell division reveals native protein localization. Nature Methods9(5), 480–2. http://doi.org/10.1038/nmeth.1955

  •            Example of fluorescent proteins changing biology in unpredictable ways.

        Mueller, F., Morisaki, T., Mazza, D., & McNally, J. G. (2012). Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis. Biophysical Journal102(7), 1656–65. http://doi.org/10.1016/j.bpj.2012.02.029

  •           Measures reversible photoswitching after FRAP for several FPs and proposes a strategy for correction

        Norris, S. R., Núñez, M. F., & Verhey, K. J. (2015). Influence of Fluorescent Tag on the Motility Properties of Kinesin-1 in Single-Molecule Assays. Biophysical Journal108(5), 1133–1143. http://doi.org/10.1016/j.bpj.2015.01.031

  •            Reports the effect of a variety of fluorescent proteins on kinesin motility. Different fluorescent proteins have significantly different (and unpredictable) effects. 

        Schnell, U., Dijk, F., Sjollema, K. A., & Giepmans, B. N. G. (2012). Immunolabeling artifacts and the need for live-cell imaging. Nature Methods9(2), 152–8. http://doi.org/10.1038/nmeth.1855

        Stadler, C., Rexhepaj, E., Singan, V. R., Murphy, R. F., Pepperkok, R., Uhlén, M., … Lundberg, E. (2013). Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells, 10(4). http://doi.org/10.1038/NMETH.2377

  •            The two papers above (Schnell et al and Stadler et al) make a nice pair to emphasize the need to validate localization data with more than one technique. Schnell et al in particular provides an excellent summary of the potential pitfalls in immunofluorescence sample prep. 

        Shaner, N. C. (2014). Fluorescent proteins for quantitative microscopy. Important properties and practical evaluation. Methods in Cell Biology123, 95–111. http://doi.org/10.1016/B978-0-12-420138-5.00006-9

  •             Excellent review of FP properties; protocol for testing FPs in your system