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Hi Subbu,
In my opinion, it is worth your while to get a better handle on the
complex before you get going with the crystallization attempts.
It's not clear to me if you are mixing the two proteins along with the
precipitant in the crystallization drop. If that is so, why not first
form the complex.
What is the numerical value for 'tight' in your case? How did you
measure that? What do you mean by precipitation in gel filtration?
First step would be to try and purify a stable complex over gel
filtration. Does the complex elute where you expect it to? Is it forming
oligomers (homo, hetero)? Is it eluting in the void volume? If so, check
the buffer components - play with each and every component to see if
that allows you to make a stable complex. Read the recent ccp4bb thread
on aggregation/solubility with respect to what all to try.
If one is running SDS-PAGE, immaterial of whether one has a heterodimer
or a supra-oligomer of the heterodimer or a total mess in solution, one
can expect to see two 'nice' bands corresponding to the two proteins in
the denaturing gel. How does that help? Try and analyze the complex by
sedimentation equilibrium or SEC-LS or DLS analysis. All of this
information will only help you understand what it is that you have in
your hands.
Your problem is perhaps that of an ill-behaved complex, which is a step
much ahead of crystallization. Try and fix this first.
HTH,
Raji
N. Ramasubbu wrote:
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Hi all:
I am trying to crystallize a complex of two proteins by mixing equal
concentration of the two.
At equimolar ratio or at 10mg/mL, for example, I get a precipitate
right away after mixing.
These two form a tight complex and even in other experiments using gel
filtration we have seen precipiatation
and when run on a gel, we see two bands with appropriate molecular
masses.
Molecular weight of one is around 56 kDa and the other one is 20kDa.
I would like to know of methods that one could try to keep the complex
in solution so that it can be crystallized.
Both are highly soluble proteins and can be crystallized individually
with out any difficulty.
Both form crystals using MPD at about 40%. Any help would be realy
appreciated.
Thanks a lot
subbu
--
Raji Edayathumangalam
Postdoctoral Fellow
The Rockefeller University
Box 224. 1230 York Avenue
New York, NY 10021
