[RASMB] Help With Understanding AUC SE Result

[Walter Stafford]

Hi Marcel,

	It looks like your speeds are too low (no appreciable curvature, which you need for resolution) and also the loading concentrations are low. But increasing the speed will help a lot. You should aim for sigmas in the range of 2-5 cm^-2.

Walter


==============================================
Walter F. Stafford, Ph.D.
Senior Scientist and
Director Analytical Ultracentrfugation Research Laboratory
Boston Biomedical Research Institute
64 Grove Street
Watertown, MA 02472
617-658-7808
skype: w.stafford3
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"Be kind, for everyone you meet is fighting a hard battle."  -Plato
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On Dec 19, 2011, at 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
> <se_profiles.jpg><sv_result.jpg>_______________________________________________
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