I am trying to sort cells on the basis of whether a fusion protein is diffuse in the nucleus of the cells or immobilized on certain chromsomal structures. When generated using GFP fusion proteins, the cells are easy to distinguish visually- either you see 48 spots corresponding to the human chromosome structure (with little background fluorescence), or you see diffuse fluorescence in the nucleus. I would like to scale the experiment up to flow cytometry throughput levels. I am hoping to find a fluorescent protein for use as the tag (e.g., to produce a fusion protein) which would have a substantial difference in anisotropy or lifetime under these conditions -diffuse tag versus immobilized tag, the latter presumably tumbling on a slower time scale. I expect that there are about 50 fusion protein molecules per spot. Any suggestions? Thank-you Matt Vincent
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