klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Tue Aug 7 20:04:52 EST 2001
"Dr. Artem Evdokimov" <eudokima at mail.ncifcrf.gov> wrote:
>And if you intend to crystallize the protein, don't do this. Even
>ammonium sulphate precipitation may not be safe, in fact.
Sure. But in this case AS precipitation is already given.
Like you yourself frequently notice, there are no hard rules with
proteins. It all depends, needs to be kept in mind and needs
to be tested as the work suits. One of the proteins I worked with,
145K, did not change its crystallization pattern +/- AS pptation (xtals
did not diffract in any case :-)), another, 38K, would not crystallize
after dialysis against pH 6.5 - even though this pH and salt
concentration kept protein active and soluble. On another extreme
is a case where acetone pption of 32K protein in the end produced
xtals scattering to 2.3A in area detector.
P.S. A question: protein from inclusion bodies spontaneopusly
misfolds into something 100% inactive but 100% soluble. What is the
best way to see if resulting sup is homogenous or not? More
filosophical Q: what are the chances of this conformation being
relevant to anything? (In other words, did Nature missed it or went
for a trouble of using alternative that does not fold spontaneously?)
>> Usially 12K proteins are quite simple and withstand quite harch
>> treatments. If yours is such a case, an attractive possibility can be
>> organic solvent precipitation. Say, you have your pellet after AmSO4
>> cut, you resuspend it in water/buffer, then add enough acetone
>> (isopropanol, dioxane or whatever works best keeping protein alive),
>> your protein precipitates and you resuspend it in whatever you like.
>> The advantage to dialysis is speed (fast) and keeping concentration
>> high. Works only for select proteins, _usually_ small.
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