[RASMB] how large a species is practical?

[John Philo]

Joel,

To me it sounds like no big problem at all. I've run adenovirus samples
(~150 MDa, see example on our web site) and even highly aggregated viruses
(~10 GDa). I've also looked at vaccines that were very, very heterogeneous.

It is just a matter of spinning slow enough to be able to "see" the largest
species (keeping them in the cell for at least a few scans). I've run my
XL-A as low as 1200 rpm (but it is happier at 3000 and higher). 

The correct rotor speed also depends of course on the vbar---things with
densities closer to water than proteins can be spun much faster.

Samples with really broad ranges of sizes of course require an appropriate
analysis method. Methods that require a plateau is still present like
standard dc/dt or van Holde-Weischet do not work well, but Peter Schuck's
c(s) or Walt Stafford's 'gravity sweep' dc/dt method should work.

John Philo
Alliance Protein Labs

-----Original Message-----
From: rasmb-admin at rasmb-email.bbri.org
[mailto:rasmb-admin at rasmb-email.bbri.org] On Behalf Of Joel Mackay
Sent: Tuesday, March 09, 2004 2:47 PM
To: rasmb at rasmb-email.bbri.org
Subject: [RASMB] how large a species is practical?


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hi all
I have had an industry chap come to me and want to explore the possibility 
of using the AU as a means to check the quality and reproducibility of 
preparations of an adjuvant (a mixture of stuff including saponins and 
cholesterol that is added to antigens when injecting them - they apparently 
help to promote an immune response to the antigen). The adjuvant forms 
aggregates that appear to be of the order of 1 MDa - too big to be retained 
on a sizing column.
My question is: am i likely to be able to record velocity data on such a 
species. I realise that a quantitative analysis of the data may not be 
feasible if these aggregates exhibit a wide range of sizes, but it may 
simply be enough to discern differences between different preparations, for 
his purposes.

Have any of you worked on such large things before, and/or on heterogeneous 
systems such as these? Any thoughts would be appreciated. cheers joel



*****************************************************************
Dr Joel Mackay
NHMRC Senior Research Fellow
School of Molecular and Microbial Sciences
Building G08
University of Sydney,
Sydney, NSW 2006
Australia
ph +61-2-9351-3906
fax +61-2-9351-4726
WWW: http://www.mmb.usyd.edu.au/mackay/
Sydney Protein Group Website: http://www.biochem.usyd.edu.au/spg/
***************************************************************** 

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