From: Les Hicks <hicks@gpu.srv.ualberta.ca>
  To  : RASMB Bulletin Board <rasmb@alpha.bbri.org>
  Date: Mon, 15 May 2000 10:14:03 -0600

Responses to highly concentrated sample query

    Many thanks to everyone who responded to my query regarding
sedimentation equilibrium of highly concentrated protein solutions.  Their
willingness to share their extensive knowledge and experience is what makes
this bulletin board such a valuable resource.  For the benefit of other
users who are interested in the same application, here are some of the
replies that might not have been posted on the bulletin board.

Cheers,

Les Hicks



>From Tom Laue :

Hi Les-
Yes, use the IF optics at low rotor speeds with several increments up to
higher rotor speeds. Use a wide dilution range, and co-fit the high and low
concentration data (taken at different rotor speeds) and you will be able
to characterize the protein very well. If this is an NMR sample (as I
suspect), it will likely be at low pH and low salt. Be ready for
nonideality. At the highest protein concentrations, you may not be able to
get an adequate fit with a single nonideality coefficient. In any case, you
will be able to answer your question.
Best wishes,
Tom




>From Les Holladay :

Hi Les:

ages ago Allen Minton and Marc Lewis did this by making a very thin
centerpiece out of the red polyethylene gasket material.  I do not have the
reference handy, but they did myoglobin this way, and showed a very slight
dimer formation.  One may have a tool and die maker actually make a tool to
punch it out.  I'm sure they will get back to you one this.

cheers

Les





>From Neil Errington :

Dear Les

at the risk of sounding heretical there is one way to deal with this
solution which doesn't have the associated problems. Schlieren optics would
answer your question easily. Unfortunately our lab seems to be one of the
few left who actually care about schlieren optics, never mind continuing to
use it. I appreciate that the XL-i doesn't have schlieren optics, but if you
found someone who was still running a Model E set up in that manner I'm sure
they would love to help. We are a little far away from you to be of
practical assistance, but we routinely analyse molecules at this kind of
solution concentration for people who want to know what their sample is
doing at NMR-type concentrations etc.

If you can't find anyone closer to home, or a different way of doing this
then get back in touch and we'll see if we can help. We have an on-line
computer capture and analysis package running on a Model E referenced first
in Clewlow et al, European Biophysical Journal, (1997).

Hope you find a solution to the problem somehow

Best wishes

Neil Errington




>From Marc Lewis :

Dear Les:

There is a very simple answer for your problem.  Some years ago Allen
Minton and I used centerpieces made of the 0.1 mm thick red polyethylene
gasket mateial that Beckman used.  This will give you gradients equivalent
to those of a solution of about 0.42 mg/ml.  I had a punch made for making
these centerpieces but have misplaced it.  However, Preson Hensley had our
machinist make one for him too and if you contact him he might make some
centerpieces for you.  If you do this I can give you specific instructions
for their use.  We found that they worked quite well.

Regards -

Marc Lewis