[Sorting on diffuse vs punctate?]

mpv@activecontact.com mpv at activecontact.com
Fri Mar 22 10:17:52 EST 2002


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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