I am performing FRAP in live cells to check the dynamics of my interested protein tagged with GFP. Its DNA binding protein. But, when I expose the sample, the entire area bleaches even if I point a small area. I am not finding this difficulty when I do it on same cells when they are fixed. What does this tell you?
If you work with living cells, the bleached area is larger than the illuminated area? Maybe it is because fast mobility of your protein - bleached proteins diffuse out of the illuminated area and other protein molecules which enter get bleached as well and so on. If the illumination lasts for long enough time, all available molecules will get bleached. How long is the illumination pulse you are using and how large the area?
Why not to use different technique? Use Fluorescence Correlation Spectroscopy (FCS). You can study dynamics at a molecular level, without damaging your cell.
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