Dear All:
Recently I am pursuing the crystallziation of a complex formd by two
individual proteins and I met several interesting problems though they are
kind of off-topic.
Any suggestions for these problems will be highly appreciated.
BIAcore showed about submicromolar affinity(both Kinetic and
steady-state fitting) for these two proteins in the complex. However,
precipitates immediately appeared when these two proteins were mixed together
even at 10uM(<0.3mg/ml) concentration in the condition of low salt(less than
20mM NaCl).
By the way, these two proteins completely precipitated when the molar ratio is
1:1 in this condition.
THerefore, I increased the salt concentraion step by step and finally I can
keep both of them soluble in the solution with 25mM Tris(pH8) and 60mM NaCl(the
minimum of salt concentration). Wierd thing happened when ITC experiments
were carried out to confirm the binding affinity. 20uM in the sample cell and
200uM in the syringe could not give enough heat for a good curve fitting. The
optimistic estimation of the affinity is lower than 5uM, which is much lower
than the affinity given by BIAcore in the same buffer(25mM Tris plus 150mM
NaCl).
Now I am suspecting the capability of the interaction between these two
proteins. However, I can not explain why these two guys precipitated
stoichiometrically if they do not interact with each other.
Is the complex salt-sensitive therefore there was just minor binding in
the high-salt condition revealed by ITC?
I am planning to do the ITC again in the condition of 25mMTris and 60mM
NaCl.
What if the affinity given by ITC is still much lower than that by
BIAcore. Which one should I choose to believe?
Are there some better ways that I can validate the binding affinity?
Thanks again for your great ideas.
Jerry McCully
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