Re: Green fluorescent protein/fixation

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From:<> (Karen Larison) (by way of Marvin Hanna)
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If I weren't concerned about retaining the fluorescence of GFP, and my goal
was to
optimize the signal from an antibody to GFP, I would start with an array of
and find out what works best.  Since a ployclonal binds to a variety of
epitopes, it
should work with a variety of different fixation methods.  But one of the
epitope-specific antibodies in the polyclonal may dominate, and the epitope
for this
dominant antibody may better be exposed with one method of fixation over
The antibodies in the brew that bind to conformation-specific epitopes will
crosslinkers to preserve antigenicity.  I have a monoclonal that I
sometimes use that
only works with Carnoy's, methacarn, or a TCA fix.  My presumption is that the
epitope for this antibody is buried, and protein extraction (Carnoy's,
methacarn) or
protein cleavage (TCA) is required to unmask the epitope.

Karen in Oregon

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