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

[Arthur Rowe]

Hi Titus
That's interesting. A 600 kDa species will be seriously distinct from  
Ficoll 70 in its sedimentation properties, even allowing for the lower  
vbar value of the latter. Which is good news.  My own approach would be  
to do an initial SV run, with solvent including Ficoll in the reference  
channel, and see if a c(s) profile reveals any (probably rather broad)  
peak in the region of 20S ( s,20,w). If so, then an SE run will  
certainly give you an average (apparent) MW value - but how to correct  
this value for the crowding effect, which will affect D and hence M?  
It's dirty, but maybe using a 'calibration' molecule (apoferritin comes  
to mind) of similar size to give a 'correction factor' could be don.   
Other technologies do not offer easy options, either. SEC-MALLS with  
columns equilibrated with 10 mg/ml Ficoll?!  DLS to give an rH - sounds  
better, but unlikely to be very precise. Anyone out there got some  
better ideas, please?
Best wishes to you and all
Arthur

On Aug 2, 2011, at 19:31, Titus M. Franzmann wrote:

> 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.
> Best
> Titus
>
> 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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>   
> *********************************************************************** 
> ********
>  Arthur J Rowe
>  Professor of Biomolecular Technology / Director NCMH Business Centre
>  School of Biosciences
>  University of Nottingham
>  Sutton Bonington
>  Leics LE12 5RD
>
>  TEL:  0115 9516156
>
>   
> *********************************************************************** 
> ********
>
************************************************************************ 
*******
Arthur J Rowe
Professor of Biomolecular Technology / Director NCMH Business Centre
School of Biosciences
University of Nottingham
Sutton Bonington
Leics LE12 5RD

TEL:  0115 9516156

************************************************************************ 
*******
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