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Eukaryotic cells regulate protein function by controlling their access to sub-cellular compartments. Knowledge of the dynamic associations of proteins with sub-cellular compartments is therefore a key determinant in understanding their function. In this context, we present two complementary techniques intended for characterising protein trafficking pathways in living cells. Firstly, we introduce the FRAP-PA unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk Confocal Microscope (SDCM) as a new & robust platform to exploit the photo-convertible properties of the fluorescent protein Dendra2 (Evrogen). Using two fast trafficking proteins (UBC 9, Fibrillarin) as proof of principle, we describe step by step procedures, with emphasis on image acquisition & processing parameters, to successfully characterise Dendra2-fused proteins trafficking pathways in live cells & in real-time. Subsequently, we present novel analytical software comprised of a simple user interface which allows the user to track the fluorescence of selected points over time, and we describe in depth the steps required to process the acquired data & analyse the resultant images. The image processing stage includes red/green image identification & separation, noise filtering, background extraction, contrast stretching & temporal smoothing. Image analysis includes the construction of mean & standard deviation images, classification of cell regions & photo-conversion point approximation.
Woods, E. et al. (2013) Tracking Protein Dynamics With Photo-Convertible Dendra2 on Spinning Disk Confocal Systems. BioPIC 2013.