[RASMB] AUC in the presence of 100 mg/ml Ficoll70

Tue Aug 2 11:31:32 PDT 2011

Hi Arthur, thanks for your reply. From SV experiment in buffer we learned that we thus far deal with a ~600 kd oligomer, however the protein only weakly associates and forms a heterogeneous solution. From concentration dependent biochemical activity experiments in buffer and buffer supplemented with ficoll we assume that Ficoll stabilizes an active oligomeric species. We wanted to directly confirm that this effect is related to the formation of a greater and more homogeneous steady-state population of possibly 600 kD oligomers.BestTitus

Titus M. Franzmann
S. G. Walter Lab
Mol., Cell. and Develop. Biol. Dept.
University of Michigan
4140C Nat. Sci. Bldg
830 N. University Ave.
Ann Arbor, MI 48109-1048
titusfranzmann.wordpress.om

CC: rasmb at rasmb.bbri.org
From: arthur.rowe at nottingham.ac.uk
Subject: Re: [RASMB] AUC in the presence of 100 mg/ml Ficoll70
Date: Tue, 2 Aug 2011 18:57:31 +0100
To: tmfr at umich.edu

Hi Titus

The first thing that occurs to me is "how big is your protein/whatever, and what are you trying to find out? Also - have you tried simply putting your system into 100 mg/ml Ficol? I ask because although it is advertised (I am sure correctly) as having "low toxicity", my own experience (many years ago) with trying to fractionate muscle filaments in Ficol was that this solvent was not exactly benign towards my precious samples!

Obviously in SV the Ficol 70 will sediment, and although it will be invisible (give or take a bit of turbidity) in the u/v, you will get some very weird Johnston-Ogston effects if the s values of your system coincide at all closely with those of the Ficol. Even if your reference channel contained the 100 mg/ml Ficol, you could still have problems. SE sounds at least as problematic - and although  you would not be plagued by things such as Johnston-Ogston effects, non-ideality/crowding effects sound daunting.

Kind regards

Arthur

On Aug 2, 2011, at 14:26, Titus M. Franzmann wrote:

Hi,
I was wondering if anyone could comment on whether there is a (proper) way to perform either AUC SV (preferred) or EQ with 100 mg/ml Ficoll70 in the buffer and which would be the best way to analyze such data.
Thanks for your assistance.
Titus
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