[RASMB] Help on AUC (sedmentation velocity)

[Jacob Lebowitz]

Yu-Chia (and all),

In addition to Borries and John's comments, I would raise the concern that 
2.3M ammonium sulfate concentration will most likely lead to preferential 
hydration of a-chymotrypsin and the vbar may be affected significantly. If 
you perform SE experiments you should model the data with the 
buoyant/reduced Mw since you will not know the vbar under your experimental 
conditions.

Jack


At 07:14 PM 4/11/2005 -0400, Yu-Chia Cheng wrote:
>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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