[RASMB] Help on AUC (sedmentation velocity)

Borries Demeler demeler at biochem.uthscsa.edu
Mon Apr 11 21:34:00 PDT 2005


Hi Yu-Chia,

I have some comment on the shifting meniscus you are describing.
We have noticed that on some machines the slit assembly tends to 
become sticky or dirty and doesn't move smoothly. This results
in shifting radial readings (only the meniscus is really obvious,
but all your readings could be affected).

On the other issues: I am unsure of the format of the attached data, so
I can't read it. Perhaps you could resend it as a PNG image or attach
the experimental data directly as a zip archive. A sigmoidal boundary
shape seems normal to me, maybe I misunderstand your description.

Regards, -Borries


> Hello,
> 
> I want to verify whether a-chymotrypsin (MW=25 KDa) dimerizes at pH 4 and
> 2.0 m and 2.3m ammonium sulfate concentration.
> Sedimentation velocity experiments were performed at 40,000 rpm and 20 deg C
> for 6 hours.
> Protein concentrations were ~3 mg/ml and absorbance at 280 nm was measured.
> 
> After plotting the raw data, I found that the meniscus at each time moved
> slightly and the movement was not a function of time.
> Also, after passing the meniscus there was a shift on the absorbance and
> then it was followed by a S-like trend endding with the upper plateau.
> There was no leaking observed at the end of experiments.
> However, due to the above behavior analyzing results becomes difficult.
> 
> Since I am new to AUC, does anyone know why this is happening?
> Could it be because the density of ammonium sulfate solution is very high so
> that higher rotating speed needs to be applied?
> Or is this Johnston-Ogston effect?
> What can I do to better experimental results?
> 
> Thank you very much for your feedback.
> 
> Sincerely,
> Yu-Chia Cheng



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