From: John Philo <jphilo@mailway.com>
  To  : Les Hicks <hicks@gpu.srv.ualberta.ca>
  Date: Sun, 14 May 2000 13:36:56 -0700

RE: Sedimentation Equilibrium of highly concentrated protein solutions

Les,

FYI by using the 3 mm centerpiece and interference optics I was able to do
velocity runs at 42 mg/ml, so 50 mg/ml in equilibrium may be possible, but I
think barely. The steepest gradients in those experiments hit about 30
fringes/mm, and I think that is pretty close to the maximum the optics can
track.

One problem, though, in equilibrium with interference data is that in order
to get a reasonable estimate of the zero offset you really need to get the
meniscus depleted. If you do that at 50 mg/ml then you've got over 50
fringes of signal so that still means a pretty steep gradient at the base.
If your sample is robust you may be better off to use a longer column and
lower speed to spread the gradient over more radial distance and just wait
longer to achieve equilibrium.

Yes, you are right that with absorbance the steep sample gradient bends the
light beam too much and for any wavelength at the steepest parts of the
gradient the apparent absorbance goes jumping up > 2. With the 3 mm
pathlength I think you may be able to do 50 mg/ml if you keep the speed very
low such that you really only deplete the meniscus maybe by 25% or so. You
won't get any curvature in the data but that's not really a problem for
determining an average mass from absorbance data provided you realize that
you must keep the offset fixed (don't float it).

John Philo
Alliance Protein Laboratories
www.ap-lab.com

-----Original Message-----
From: Les Hicks [hicks@gpu.srv.ualberta.ca]">mailto:hicks@gpu.srv.ualberta.ca]
Sent: Friday, May 12, 2000 3:36 PM
To: RASMB Bulletin Board
Subject: Sedimentation Equilibrium of highly concentrated protein
solutions


Hi all,

    At the risk of asking a stupid question, here goes.  Is there any way to
perform a sedmentation equilibrium run on an approximately 50 mg/ml protein
solution?  Even with a 3 mm path length cell, I would think the fringes
would be too compressed at this concentration.  I was initially wondering
about using the absorbance optics at a higher wavelength (ie : where the OD
is app. 0.5) till I re-read Tom Laue's article "Choosing which optical
system of the Optima XL-I Analytical Ultracentrifuge to use".  I gather that
a problem would arise with an effective misalignment of the optics due to
the large refractive index gradient from the high protein concentration.
    If I used the interference optics at a relatively low speed would I
possibly be able to resolve the fringes to the cell bottom or would there be
the same problem with the interference optics due to the high refractive
index gradient?  We don't require an accurate Kd for this sample - we would
just like to know whether it's mainly monomeric, dimeric, or whatever at
this high concentration.

Regards, and have a good week-end.

Les



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Les Hicks
PENCE / Dept. of Biochemistry
Rm 3-36 Medical Sciences Bldg.
University of Alberta
Edmonton, Alberta, Canada
T6G 2H7

Phone : 780-492-3412
Cell     : 780-975-7741
FAX    : 780-492-0095
email   : hicks@gpu.srv.ualberta.ca
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