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CCP4BB Home

CCP4BB  January 2008

CCP4BB January 2008

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Subject:

Re: Web site GOOD, Attachment BAD (was: salt sensitive complex)

From:

Raji Edayathumangalam <[log in to unmask]>

Reply-To:

[log in to unmask]

Date:

Thu, 31 Jan 2008 08:12:33 -0500

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (158 lines)

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 &#177; 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     &#177;0.17
>>>     >
>>>     > K         1.49E5 &#177;1.5E5
>>>     >
>>>     > DH       -893.5  &#177;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>
>>
>
>
---------End of Included Message----------

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