From: Disher, Rose [CNTUS] <DisherR@Centocor.com>
  To  : 'Les Hicks' <hicks@gpu.srv.ualberta.ca>
  Date: Wed, 17 May 2000 09:04:36 -0400

RE: Responses to highly concentrated sample query

This has been an interesting and enlightening discussion.  The modified cell
construction is also of interest to those of us in the industrial community
looking at what happens to our products in process and in formulations.  It
is not unusual for a biopharmaceutical to spend time at concentrations in
the 30+ mg/ml range, and we sometimes have to look at high concentrations to
answer questions for the regulatory agencies.  I have used the 3 mm
centerpieces in sedimentation velocity studies, and it isn't difficult to
imagine needing to get 1 mm cells at some point in the future.  Does anyone
know if Beckman or anyone else would do a special order for something like
this for those of us who don't have ready access to an appropriate machine
shop?  (If any Beckman folks are reading this, consider it a product hint.)
I am interested in getting a contact and a lead time in case I need to get
some really thin cells for my studies, and there may be some others
interested in this type of information as well.

		-----Original Message-----
		From:	Les Hicks [hicks@gpu.srv.ualberta.ca]">mailto:hicks@gpu.srv.ualberta.ca]
		Sent:	Monday, May 15, 2000 12:14 PM
		To:	RASMB Bulletin Board
		Subject:	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