[RASMB] Help With Understanding AUC SE Result

Walter F. Stafford stafford at bbri.org
Mon Dec 19 14:36:03 PST 2011


Marcel,

     You can compute the time to equilibrium using the equation of Williams et
al (1958). You can find a little widget here that will do it for you:

http://www.bbri.org/faculty/stafford/time_to_equilibrium-form.html

But in the end, I would advise using WinMatch to verify that your system
really is at equilibrium. Several factors can influence the actual time to
equilibrium including non-ideality and slow kinetics. WinMatch can be found at
the University of Conn. site as well as the software archive on the RASMB
site.

Best wishes,
Walter

======================================
Walter F. Stafford, Ph.D.
Senior Scientist
Boston Biomedical Research Institute
tel: 617-658-7808
skype: w.stafford3

"Be kind, for everyone you meet is fighting a hard battle."  -Plato
------------------------------------

On Mon, December 19, 2011 06:31, Marcel Jurk wrote:
> Dear all,
>
> Recently, I performed an AUC SE experiment that resulted in "strange"
> concentration profiles. SE is not a method I am using routinely so I am
> lacking experience to understand what might have gone wrong. I would be
> grateful for any advice or help with interpretation.
>
> But first things first. The monomer of the target protein is 12 kDa. Sample
> volume was 80 µL at a temperature of 25 °C. Samples were kept at RT for 5
> days after dilution and before the run. Buffer is PBS (~150 mM NaCl; ~20 mM
> K/Na-P; pH 7.4). SDS-PAGE after centrifugation showed no (detectable) protein
> degradation.
>
> The samples were spun 36h at 21k rpm. After this period little sedimentation
> had occurred. Increasing the speed afterwards to 37k rpm for another 24h
> resulted in concentration profiles only looking slightly different.
> I attached a picture showing scans at 21k after 36h ("se_profiles.jpg"; top)
> and 37k after additional 24h ("se_profiles.jpg; bottom").
>
> Is it worthwhile doing another run but keeping the protein initially for a
> prolonged time (>>5 days) at experiment temperature (25 °C)? Or is 37k rpm
> still too slow?
> The idea for doing an SE experiment came up because SV runs at 45k indicate a
> dimerization of the protein. So 45k was okay for SV (see "sv_result.jpg").
>
> Any advice would be helpful.
>
> Best regards,
> Marcel
>
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