[RASMB] High sucrose concentrations in sed velocity experiments

[David Scott]

Hello Matt,

Basically, small solutes will form a dynamic density gradient when you perform sedimentation velocity. If there are enough around, then you have to correct for it, which appears to be what you have here. We have a similar problem as we work with extreme halophilic proteins that are happy only in molar salt concentrations. Luckily Peter Schuck has written a nice routine in SEDFIT to correct for this (under options, select inhomogenous gradient, click "no" to compressible solvent and "yes" to dynamic gradient...). It takes longer for everything to fit, but gets there in the end. 

The full and gory details are in a Biophys. Chem. paper:

P. Schuck (2003) A model for sedimentation in inhomogeneous media. I. Dynamic density gradients from sedimenting co-solutes.  Biophysical Chemistry 108:187-200

and further details of its implementation in SEDFIT are on Peter's website under:

http://www.analyticalultracentrifugation.com/dynamic_density_gradients.htm


Have fun!

Dave Scott.





Dr. David J. Scott
Lecturer in Physical Biochemistry
National Centre for Macromolecular Hydrodynamics
School of Biosciences
University of Nottingham
Sutton Bonington
Leicestershire
LE12 5RD
Phone: +44 (0) 115 951 6221
Fax: +44 (0) 115 951 6142
Email: dj.scott at nottingham.ac.uk
http://www.nottingham.ac.uk/biosciences/foodsci/academic/scott.html 

>>> "Parker, Matthew" <Matthew.Parker at immunogen.com> 11/14/05 3:24 pm >>>
            Hi. We recently ran sedimentation velocity on some samples that
contained 10% sucrose in the buffer. We obtained results for the c(s)
distribution that didn't agree well with what we saw by SEC, and the s-values
were also shifted to significantly lower values than what we would expect
(and that we have seen in other formulations) for an IgG. It was suggested to
us that our sucrose concentration was high enough to create a sucrose
gradient in the cell during the course of the run, and that this could cause
the relative peak areas in the c(s) plot to be inaccurate.

 

            Does anybody have any comments about this, or suggestions about
how high in sucrose content you can go before this starts to become a
problem?

 

            Thanks,

 

            Matt

 

Matthew Parker, Ph.D.

Analytical and Pharmaceutical Sciences

Immunogen, Inc.

Cambridge, MA



This message has been checked for viruses but the contents of an attachment
may still contain software viruses, which could damage your computer system:
you are advised to perform your own checks. Email communications with the
University of Nottingham may be monitored as permitted by UK legislation.