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Betreff: Re: [ccp4bb] Bypassing phase separation for nice crystals.
Datum: Mon, 18 Jul 2011 17:01:27 +0200
Von: Florian Sauer <[email protected]>
An: F. Timur Senguen <[email protected]>
CC: [email protected]
Dear Timur,
one possibility to handle this problem can be the change from vapor (I
assume this is what you do) to the free interface diffusion method.
Phase separation often occurs if the protein is immediately exposed to
the full precipitant concentration while it might not "escape" into its
own phase if it gets slowly equilibrated.
There are commercial setups available for this method but you can also
do it in a normal vapor diffusion plate.
To do so, just put the protein and precipitant drops next to each other
and then link them through a thin liquid bridge. Requires some practice
and works best with large drops but helped me in several similar cases.
Good luck!
Florian
Am 18.07.11 15:52, schrieb F. Timur Senguen:
Hi everyone,
I have been issues with a particular protein. I have been close for a
while, but yet so far.
Rather than going from a clear drop to crystal, my protein first
undergoes phase separation (large oily drops) in which one phase
contains most, if not all, of the protein. This phase separation
occurs within a day of preparing the drop. A day after phase
separation the oily phase is now a large disordered crystalline mass
which does not diffract very well. I have tried changing buffer
concentrations, precipitant amounts, ionic strengths and pH and in all
cases this phenomenon is observed. I even screened protein
concentrations to see if reducing protein concentration would prevent
the phase separation.
Is there any way to bypass this phase separation, which I think
prevents me from obtaining nice crystals. Should I try detergents,
chaotropes, or other additives?
Thanks in advance.
Timur
--
F. Timur Senguen, Ph.D.
Postdoctoral Research Fellow
Boston Biomedical Research Institute
64 Grove Street,
Watertown,
MA 02472 USA
