Can the CCP4BB provide something like a website to upload pictures and then have the BB-ers just
post the link in their email. Please!
These attachments are clogging my inbox...
Thanks much.
Raji
---------Included Message----------
>Date: 31-jan-2008 03:58:44 -0500
>From: "Frank von Delft" <[log in to unmask]>
>To: <[log in to unmask]>
>Subject: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)
>
>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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>>> <http://biggestloser.msn.com/>
>>> <test-ITC-012608.JPG>
>>
>
>
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