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[ccp4bb] FW: [ccp4bb] salt sensitive complex
 

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CCP4bb <-- 2008 <-- January 2008 <-- 30 January 2008
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Subject: 2fo-fc and fo-fc
From: JINJIN ZHANG zhang {- dot -} 508 {- at -} OSU {- dot -} EDU
Date: 2008-01-30 		Next message:
Subject: Re: FW: salt sensitive complex
From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU
Date: 2008-01-30


Subject: FW: salt sensitive complex
From: Jerry McCully for-crystallizaiton {- at -} HOTMAIL {- dot -} COM
Date: 2008-01-30


Dear All:

Thanks a lot for the prompt reply on this topic of salt sensitive complex.

Attached please find one ITC final figure done under 25mM Tris(pH8.0), 60mM NaCl.

As mentioned before, the ionization of Tris will interfere with the ITC experiments.

Therefore I am sure of my binding results.

Can anyone give me some comments on this ITC experiment? Basically do these two proteins bind to each other? If so, how should I improve the ITC experiments to get a similar affinity shown by BIAcore(about 0.5uM)?

Thanks again.

Jerry

Hi Jerry,

Tris can cause problems, you are better off using something like
HEPES, and HEPES should be ok at pH 8. (Buffers with an
ethane-sulphonic acid group tend to be the best - those ending in
'ES', so MES, TES and HEPES)

FYI, the error on your K is bigger than the actual measurement -
1.49x10^5 ± 1.5x10^5.
Signal to noise to is probably your enemy, which is making the curve
fitting difficult. Changing buffer may help this - there may be some
non-specific component to what you're seeing - increasing salt a bit
or dropping in something like 5% glycerol may help with this.

Would you be able to post a jpeg/pdf of the curve?

Regards,

David


On 25/01/2008, Jerry McCully wrote:
>
> 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 20.7
>
> Was 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
>

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