From: Jo Butler <pjgb@mrc-lmb.cam.ac.uk>
  To  : Dr DJ Scott , rasmb@alpha.bbri.org
  Date: Tue, 07 Aug 2001 12:27:38 +0100

Re: Membrane proteins

Dear Dave,

Not a no-win situation at all.  We regularly look at the molecular weights 
of membrane proteins, to determine the aggregation.  The catch is that one 
really needs to measure the apparent partial specific volume (or, more 
precisely, the density increment) at "dialysis equilibrium" with the 
buffer.  This can be achieved either by the necessary dialysis or, 
sometimes more simply, by gel permeation chromatography to adjust the 
protein/detergent micelles into the detergent buffer, provided that these 
micelles can be separated from the detergent alone micelles.
With the Paar densitimeter one only needs 1ml sample, although this should 
be at a reasonably high concentration (5 - 10 mg/ml for choice).  I gave a 
talk on using this technique at the UK AUC meeting a few years ago, which 
was published in the report on the meeting (Butler, P. J. G. (1998). Use of 
density increment in assessing protein aggregation under unusual 
conditions. Biochem. Soc. Trans. 26, 749-753).

This method has the advantage of not requiring a special detergent or 
detergent mix, but being applicable to the detergent in which your membrane 
protein is most stable, or is isolated.

Good luck,

Jo

--On Tuesday, August 7, 2001 11:43 am +0100 Dr DJ Scott <djs17@york.ac.uk> 
wrote:

> Hello all
> I wish to do AUC on a (possibly) tetrameric membrance protein. The catch
> is that the detergent has to be at a conc around the CMC so there will be
> lots of micelles there.
>
> Can AUC give any Mwt or shape info. in these circumstances? I seem to
> remember previous emails on the subject of density matching, or am I in a
> no-win situation?
>
> Thanks in advance
>
> Dave Scott.
>
> Dr. David J. Scott
> York Structural Biology Laboratory
> Department of Chemistry
> University of York
> YORK. North Yorkshire. Y010 5DD
> Phone: +44 1904 432590
> Fax:   +44 1904 410519
> Email: djs17@york.ac.uk
> Home Page: www-users.york.ac.uk/~djs17
>
> PLEASE NOTE: FROM 1ST SEPT NEW ADDRESS: DEPT. BIOCHEMISTRY, SCHOOL OF
> MEDICAL SCIENCES, UNIVERSITY OF BRISTOL, UNIVERSITY WALK, BRISTOL. BS8
> 1QD. UNITED KINGDOM.
>



P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296