[RASMB] newbie to ultracentrifugation

[odonnell at chem.fsu.edu]

Hello,

I'm new to the technique of ultracentrifugation and would like to use  
it as a troubleshooting step to purify my sample. I was hoping someone  
could direct me to a good source for introductory  
information/principles.

To the reader who has generous time for advice:

Specifically, i am trying to purify a large marcromolecular complex  
from background proteins and possibly different complex types. This  
yields a range of molecular weights species that can be separated. The  
desired complex requires a high salt concentration ~700 mM KOAc for  
formation  so stability issues arise with varying ionic strengths. So  
i'm thinking a CsCl gradient is an inferior approach to a sucrose  
gradient??? So far i've done some practice runs (without any sample)  
of step sucrose gradients from 0-60% sucrose at 10% and 15% increments  
at 25K RPM on a SW28 rotor ("slow" accell and decel profiles)  with  
mild success at observing the density fractions after the spin.  
Various questions that i'm  trying to find a good source to help  
answer (besides RASMB!) are below:

Is the high salt (700m KOAc) is a problem for my density centrifugation?
Is there an upper limit to the speed that I can spin (besides a limit  
imposed by rotor specs)?
Is there a formula that helps derive the required density range to  
crate in the graient for separating a particular molecular weight  
species.

Obviously, any advice is  greatly appreciated!!

-Jason

----------------------------------------------------------------
This message was sent using IMP, the Internet Messaging Program.



-- 
This message has been scanned for viruses and
dangerous content by MailScanner, and is
believed to be clean.