Dear Min,
Regarding the stoichiometry that you should use in crystallizing two
proteins
that form a complex. I have looked at this question before. See:
Stura, E.A., Graille, M., Taussig, M.J., Sutton, B.J. Gore, M.G.,
Silverman, G.J., Charbonnier, J.-B. (2001)
Crystallization of macromolecular complexes: Stoichiometric variation
screening. J. Cryst. Growth 232:580-590.
http://www.sciencedirect.com/science/article/pii/S0022024801011721
Briefly: The stoichiometry can, and should, be varied in your screening.
The relative protein solubility of the two individual proteins and the
solubility of the complex should be
analysed under various potential crystallization conditions. Conditions
where the complex is less soluble
than the individual ptoteins should be chosen if the complex has a
tendency to dissociate.
Since both proteins have been crystallized before you may also use
crystals of both the free forms of the two proteins to stimulate
nucleation of the complex
The final composition of the asymmetric unit may include free poteins as
well as the complex.
The paper will give you a lot more methodology to use to obtain crystals
of the complex, if
such complex really exists, is homegeneous and relatively stable.
Enrico.
On Fri, 16 Sep 2011 04:20:26 +0200, m zhang <mzhang...@hotmail.com> wrote:
Dear all,
I have two questions:
First, I was trying to crystallize a complex of two proteins. Both
proteins has been crystallized before. The two proteins bind to each
other based on Biacore study, but they didn't form a single peak on gel
filtration. When I mixed them at 1:1 ratio, the crystals I got contain
only one of the two proteins. I was suggested to increase the ratio, for
example 1.5:1, to increase the probability of co-crystallization which I
will try. But I do want to hear if there are other possible ways to try.
What would you try if you were in my situation?
Second is about reusing of Ni-NTA resin. According to Qiagen's
instruction, after using fresh Ni-NTA resin, one only needs to wash the
used Ni resin first with 0.5M NaOH, then with your own buffer. After
that the resin is ready to be reused until it needs being recharged. But
my question is: Once immidazole competes with His-tagged protein and
binds to Ni-resin, how can immidazole be rinsed off with the same
buffer(usually pH is above 7) one uses to purify the protein?
Thank you for any suggestion or comment.
Min
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
