[RASMB] Help on AUC (sedmentation velocity)

[Yu-Chia Cheng]

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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