Actually, it's not book keeping, it's simple courtesy -- and not only on
a BB: an attachment is lazy, and a large attachment is downright rude.
I am routinely stuck with a slow connection (travelling), and others are
*permanently* stuck with one. So please be nice... ;)
phx.
Anastassis Perrakis wrote:
Dear all -
Sorry to intervene on a 'book keeping' issue, but indeed over the last
few months an increasing number of people (Jerry is not the first, so
Jerry please do not take it personally) attach pictures etc. I think
in a bb standard practice dictates to only use text - if illustrations
are needed to explain the problem, you can put them in eg a web site.
Some text like that was in the 'code of conduct' off ccp4bb in the
past, but I could no longer find it.
Thus apologies if I am wrong and policies have changed, but maybe the
ccp4 crowd could tell us what is the suggested policy.
And, if you really want to send an image please do bother to make it
small. The initial posting had a 630k image, which it took me 1 min to
make 20k and it still makes the point (attached so I can also violate
the rules i am suggesting - I love inconsistency).
Thanks, Tassos
On Jan 30, 2008, at 20:11, Jerry McCully wrote:
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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