Re: [ccp4bb] FW: [ccp4bb] salt sensitive complex

[Anastassis Perrakis]

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. Shed those extra pounds with MSN and The Biggest Loser! Learn more. <test-ITC-012608.JPG>