Re: Freezing fluorophores

Posted by Martin Wessendorf-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Freezing-fluorophores-tp591414p591419.html

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Fredrik Wermeling wrote:

> What’s your opinion about freezing fluorescently conjugated antibodies?
> I know for example that the Qdots are destroyed by freezing, but how
> about others?

Dear Fredrik--

In my experience, freezing is almost always bad.  The signal-to-noise
ratio of the secondary antibodies that I use (C2, Cy3 and Cy5
conjugates) goes from being superb to being poor with one freeze-thaw
cycle.

The problem with freezing appears to be denaturation of the IgG to which
they're conjugated.  The explanation that I've heard is that as an
antibody solution freezes, the pure water crystallizes first.  What
remains has a higher concentration of salts.  As freezing progresses,
the concentration of salt in the liquid fraction becomes higher and
higher, ultimately resulting in denaturation of the protein present in
that fraction.

By that logic, if you need to freeze, very rapid freezing should be
better than slow freezing.

As an alternative to freezing, you can store your antibodies at -20 C
without freezing by diluting them 1:1 in glycerol.  (That trick comes
from Bill Stegeman at Jackson.)  In my experience that works well.

Good luck!

Martin Wessendorf
--
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