[RASMB] Help With Understanding AUC SE Result

[Borries Demeler]

Marcel,

a couple of points:

I would increase the column length to get more signal. The data you
show is actually quite noisy, which is a problem if you have so little
signal. Instead of loading .08 ml I would load 1.2 ml. If the monomer
sediments at 1.8s, then the monomer is almost spherical and has a large
diffusion coefficient. The UltraScan equilibrium time simulation predicts
that you should run between 30-55 krpm.  If you expect to see some dimer,
you could reduce the speed a little and increase the time. Check the
UltraScan Equlibrium Time simulator. Due to the fast diffusion of this
protein, you can expect faster equilibration than you used before:

RPM:  Time Increment:   Total Time:

30000   15.50 hours     15.50 hours
38300   11.75 hours     27.25 hours
46600    9.00 hours     36.25 hours
54900    6.25 hours     42.50 hours

(you should add time to scan, of course)

Based on the image of your experimental data, it seems unlikely that the protein is
aggregating, it seems to be well behaved, so you should be OK with the total time
of this experiment. Buffer absorbance is NOT a problem. The buffer you list doesn't
absorb at 280 nm. Dialysis is not required for optical reasons since you aren't 
running in interference mode.

Regards, -Borries



On Mon, Dec 19, 2011 at 02:34:33PM +0100, Marcel Jurk wrote:
> Thank you, Walter and Andrew for your quick replies.
> 
> So, I will do a re-run with higher speed. Protein concentration is, unfortunately, limited (as always...).
> 
> The protein should be in dialysis equilibrium with the buffer (I demanded that and was told that is). On the other hand, there was no buffer background in SV and samples were from the same stock.
> 
> The S-value I got is ~1.8 for the monomer with a small shoulder. The latter becomes a distinct peak when increasing the concentration to max available.
> 
> It's likely that besides the self-association there is a conformational equilibrium between folded and partially unfolded but that's "only" an assumption of the colleague who's protein we are talking about.
> 
> Best,
> Marcel
> 
> Marcel Jurk
> Leibniz-Institut fuer Molekulare Pharmakologie (FMP)
> Solution NMR (AG Schmieder)
> Robert-Roessle-Str. 10
> 13125 Berlin
> Germany
> 
> eMail: jurk at fmp-berlin.de
> Phone: +49-30-94793223
> Fax: +49-30-94793169
> >>> "Leech, AP"  19.12.11 14.16 Uhr >>>
> Hi Marcel,
> 
> Have you used the equilibrium simulator in the Beckman software?
> This will give you a good idea of what to expect. Putting in your
> protein parameters seems to give more curvature than you see in
> the experimental data. This could be the case if there is some
> other component absorbing in your sample (e.g. buffer).
> 
> You don't say what the results of the SV experiment are except that
> it indicates dimerisation - is it all dimer or only part? Is the
> sedimentation coefficient reasonable?
> 
> All the best,
> 
> Andrew
> 
> On 19/12/2011 11:31, 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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> 
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