saarenma at utu.fi
Tue Apr 8 02:19:05 EST 1997
Hello, news readers!!!
I am having terrible problems with the detergent DIGITONIN.
I use it to solubilize thylakoid membranes into grana stacks and
stroma thylakoids. Everything worked perfectly when we used the
CRYSTALLIZED digitonin manufactured by Merck, but when the company stopped
producing it in this form, our problems started. We have tried all kinds
of other digitonins (Sigma, CalBiochem, Baker, Merck), but nothing seems
to work. We have also tried to crystallize these digitonins ourselves, but
the result is always the same: unsolubilized material.
Why should the crystallized detergent be more efficient? I can understand
that if during the crystallization process you get rid of impurities that
might interfere with its activity, but in our case everything gets so
easily dissolved in abs. ethanol, that I can't see any reason for
filtrating it right away when still hot...
We crystallize it as follows:
1 g digitonin in 20 ml abs. ethanol
Heated for 10-15 min until completely dissolved.
Let stand in the cold room (4 C) until crystallized (several hours or o/N)
Filtrate the crystals.
When preparing the stock solution, is it necessary to heat it up or is
it enough if the solution is just cloudy but homogeneous?
I would really appreciate any hints and comments on this subject.
I am completely lost and don't have any clue on how to continue!
Thank's a lot!
Elena Baena Gonzalez
Lab. Plant Physiology and Molecular Biology
Department of Biology
University of Turku
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