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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> >