(again) [RASMB] Help on AUC (sedimentation velocity)

[John Philo]

Also I forgot to mention that the absorbance in your experiment is MUCH too
high (it is reading it as 2.2, but that is effectively infinity). In
velocity mode (continuous scan mode) the linearity of the absorbance system
is significantly lower than for equilibrium (step scan) mode, so it is best
to stay below 1 OD. 

If you want to run the 3 mg/ml the best way to do the experiment is to use
the 3 mm pathlength 2-channel centerpieces. With a 12 mm centerpiece, it
would help a lot to use the absorbance minimum near 250 nm, but that still
might not reduce the OD sufficiently, so you might have to go off the 280
peak to 295 nm or some such. 

John

-----Original Message-----
From: rasmb-admin at server1.bbri.org [mailto:rasmb-admin at server1.bbri.org] On
Behalf Of Yu-Chia Cheng
Sent: Monday, April 11, 2005 4:14 PM
To: rasmb at server1.bbri.org
Subject: [RASMB] Help on AUC (sedmentation velocity)


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