[RASMB] Help on AUC (sedimentation velocity)

[John Philo]

Yu-Chia (and all),

Although Borries couldn't read the file (it is one from my XLGraph utility)
he still got it right, you seem to have a problem with your radial scans
that caused a sudden shift of the meniscus. You may have some dirt
underneath the slit assembly. In addition to that hardware issue, it is
likely that you would see some optical distortions near the meniscus and
perhaps at the base of the cell too from the sedimentation of the ammonium
sulfate.

But if that is all the data you took then you only completed less than 20%
of the run (the boundary only made it <20% of the way to the cell base)!
With all that ammonium sulfate present the sedimentation will be very, very
slow. I would run that experiment at 60000 rpm (actually I would do that
even without the ammonium sulfate). 

Finally, for your stated purpose you really need to be doing sedimentation
equilibrium, not sedimentation velocity. It impossible to tell whether you
have a reversible monomer-dimer system from a velocity experiment at any one
concentration, and quite difficult to prove that hypothesis even with
velocity data over a wide range of concentration. 

John Philo
Alliance Protein Laboratories

-----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