[RASMB] upper concentration limit AUC

[Arthur Rowe]

Dear Joris and all RASMBers

(1) There is a problem with using sedimentation velocity at protein
concentrations in the 10-20 mg/ml level (or greater), in that the very sharp
gradients which are formed deflect the beam out of the angular aperture of
the optics. So you end up with a shadows ('schlieren') where the boundary
information should be. Using shorter optical path length cells helps here -
although you get increased problems with optical artifacts of the 2nd or 3rd
order (see below) these cause much less problem than with SE work

(2) But I guess that your colleague would be thinking about looking at Kd
values via SE? In this case, no problem need arise using 12 mm optical path
length cells provided that you keep the sigma value down to below 1. If you
try to go for higher sigma values, then you will simply lose the trace at
the higher radial values. Contrary to what is often supposed, provided the
instrument is correctly focussed onto the 2/3rd plane of the cell (i.e. 8 mm
beyond inner side of entry window) then there are no artifacts which arise
from the 3rd order terms in the Svensson equation (which relates fringe
shifts to RI increments). BUT - do not use shorter optical path length
cells. The plane of focus now lies outside the actual fluid contents of the
cell! Heaven knows what this does the the validity of your fringe
increments.

I describing the interpretation of high concentration SE runs in some
details at the Newcastle AUC Meeting at the end of next week and in a MSS I
am submitting therefrom, but some of the background is in my chapter in the
'White Book' - "Analytical Ultracentrifugation - Techniques & Methods" (eds
Scott, Harding & Rowe RSC 2005) as well as in various publications
(available on request).

Kind regards

Arthur


Dear all, 



A colleague asked me whether analytical ultracentrifugation has an upper
limit with regard to the concentration of the protein. They want to measure
the protein at the same concentration as the NMR experiments (> 1 mM). I am
not entirely sure but I thought this should be no problem. One could easily
measure off-peak at another wavelength than 230nm, e.g. 235nm or 280nm,
right?! Or does one run into non-ideality phenomena when doing sedimentation
equilibrium at these high protein concentrations?!

Thanks a lot in advance for any feedback.

Best wishes, 



Joris Beld 



Hilvert Group 

ETH Zürich 

Switzerland 

_______________________________________________
RASMB mailing list
RASMB at rasmb.bbri.org
http://rasmb.bbri.org/mailman/listinfo/rasmb




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.

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://list.rasmb.org/pipermail/rasmb-rasmb.org/attachments/20080901/059c5910/attachment.htm>