Dear All:
Firstly I would like to thank many folks here for giving me great ideas
several days ago.
The following are some updates for this question.
I did ITC experiments again using 25mMTris(pH8), 60mM NaCl(low salt
condition).
But things still turn out to be a little weird.
I increased the concentration of both proteins(60uM in the cell and 1200uM
in the syringe). At the end of the ITC, I saw a little of precipitation of both
the proteins.
Fortunately I can roughly fit the curve this time. However, the heat was
still low, around 1Kcal/mole of per injectant. I am not sure about the fitting
statistics.
N 1.10 ±0.17
K 1.49E5 ±1.5E5
DH -893.5 ±213
DS 20.7Was the enthalpy was offset by the ionization of Tris buffer?Can I
use Hepes buffer around pH8 to do ITC?
Welcome any comments about the statistics and suggestions on how to improve
the ITC experiments.have a nice weekend.
Jerry
>
> Jerry McCully wrote:
> > 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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>
> --
> Edwin Pozharski, PhD, Assistant Professor
> University of Maryland, Baltimore
> ----------------------------------------------
> When the Way is forgotten duty and justice appear;
> Then knowledge and wisdom are born along with hypocrisy.
> When harmonious relationships dissolve then respect and devotion arise;
> When a nation falls to chaos then loyalty and patriotism are born.
> ------------------------------ / Lao Tse /
>
>
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