[RASMB] Help With Understanding AUC SE Result

Marcel Jurk jurk at fmp-berlin.de
Mon Dec 19 05:34:33 PST 2011


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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