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The distribution of many drugs in cells is affected by the action of pumps
and other enzymes, many of which are in turn dependent on ATP concentration
and/or on the maintenance of potential gradients across cytoplasmic and
mitochondrial membranes. Even without fixation, whatever you need to do to
make big enough holes in the cytoplasmic membrane to admit an anti-drug
antibody will transiently abolish potential gradients and allow small
molecules to leak out of the cell. Thus, what you'll see with confocal is
very likely to be artifactual, and fixation will only move things further in
that direction.
This doesn't mean that you won't get any information. For example, if you
had a drug that bound covalently or with very high affinity to DNA, you
should certainly be able to find it in the nucleus with a fluorescent
antibody, but any conclusions you might draw about cytoplasmic trafficking
of the same drug would probably be suspect.
-Howard
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web