We use fluorescence recovery after photobleaching (FRAP) to calculate the diffusion coefficient of GFP in the nucleoplasm of fission yeast. The FRAP method can be generally used to measure the mobility of proteins inside the cell or its organelles.
In our experiment we only measured the diffusion of GFP inside the nucleoplasm of fission yeast mitotic cells. However, if GFP is fused to a protein, the mobility of the protein of interest can be calculated following the GFP signal in the bleached area. We did not, however, address this in our experiments; therefore other sources could be searched for this topic.
To compare FRAP and FLIP, both techniques can be used to measure protein mobility inside a cell. However, with FRAP, the diffusion of a protein is measured in the region of interest (ROI), to observe the recovering of fluorescence in this area. In FLIP, fluorescence recovery is measured in an area different from where the bleaching was done, to observe whether the tagged protein is able to move into that area, which would become darker, gaining the bleached proteins. The major difference here is that for FRAP a single bleaching event is sufficient, while FLIP requires a number of bleaching steps, in order to avoid reflux of fluorescent protein in the same region.
Technically FRAP in the nucleus und FRAP in the cytoplasm has no difference. However, we measured a difference between the diffusion coefficient inside the nucleus (D = 5.6 ± 2.8 μm2/s) and in the cytoplasm (D = 8.6 ± 2.2 μm2/s). This is due to the different compositions inside these compartments, consisting of differing amounts of proteins, DNA and RNA.
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