[RASMB] Help needed

[Tom Laue]

Hi Sen-
It may be that you have both a reversible association and some 
irreversible dimer. The quantity of irreversible dimer may grow over 
time, making it appear as though the Kd is dropping. Have you tried 
using a fitting model that incorporates both reversible and irreversible 
steps?
Also, if you have irreversible dimer present, the can be fractionated 
(by size exclusion chromatography). Analysis of the leading edge of the 
peak from the SEC will have a low apparent Kd, whereas material from the 
trailing edge will have a higher apparent Kd.
Best wishes,
Tom


On 8/23/2012 9:53 AM, Sen Indrani wrote:
>   Dear RASMB members,
>     I am a PhD student at ETH Zurich and have encountered a problem in my sedimentation velocity experiment run which goes as follows:
>
> I am trying to determine the dissociation constant of a dimeric protein. I am using Fluorescence Sedimentation Velocity. I had measured the protein once immidietly after purification with 2-3hrs of incubation ( including sample preparation time ) at 22°C . At that point the protein had yielded a value of Kd = 60nM. But when I repeated the experiment with the same batch of protein afetr 6months I couldn't reproduce the result and got a tigther Kd( less than 5nM). I even made a fresh preparation of the protein and the result was the same giving a tighter Kd. The only differences in the experiments where the incubation time before start of the run. For the latter cases the protein was incubated for over 16hrs or longer. So my question is that could be that the equilibrium shifts during this incubation time. If yes , what kind of experiments can be done to make sure that the Kd for dimerization is indeed incubation time dependent. Please could you suggest any literature in this respect as well.
>
> It would be of great help if anybody could help me out. I am really struggling with this problem . With regards to the data quality , I admit there is a difference ( the recent data is better ) , but I wouldn't imagine that it would produce such dramatic effect on the properties of the protein.
>
> Looking forward to your reply,
> Please write to me if you have any further questions.
>
> Thanking you,
> Regards
> Indrani
> PhD student
>
>
>
>
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>     1. Kira Shapiro is out of the office. (Kira B Shapiro)
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> Date: Fri, 17 Aug 2012 09:14:06 -0700
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> Subject: [RASMB] Kira Shapiro is out of the office.
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> I will be out of the office starting  08/16/2012 and will not return until 08/20/2012.
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> I will respond to your message when I return.
> Thank you and have a great weekend.
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-- 
Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu