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
>> >
>>
>>
>>
>> ------------------------------------------------------------------------
>> Helping your favorite cause is as easy as instant messaging. You
>> IM, we give. Learn more.
>> <http://im.live.com/Messenger/IM/Home/?source=text_hotmail_join>
>>
>>
>> ------------------------------------------------------------------------
>> Shed those extra pounds with MSN and The Biggest Loser! Learn more.
>> <http://biggestloser.msn.com/>
>> <test-ITC-012608.JPG>
>
|