From: Katharina Spiegel <k-spiegel@nwu.edu>
  To  : RASMB@bbri.org
  Date: Tue, 24 Aug 1999 14:24:24 -0500

Density gradient centrifugation with XLA?

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Dear RASMBers:

I take care of a multi-user XLA and I was recently asked by an user whether
there is any information on  how to do a density gradient run with the XLA.
Hereby I would like to forward the more detailed explanation of the
experimental problem to this group of experts. Any advice for us?


>	We would like to examine a lipid-DNA complex by centrifugation.  We
>think
>that this complex will form at a particular stoichiometry with one or the
>other of the components being in excess.  The lipid can be labelled to
>aborb in a part of the spectrum different from DNA absorbance.  The
>particle sizes are quite variable, so velocity centrifugation will not give
>us information on composition, although it may be useful eventually for
>assessing the relationship between size and composition.  On the other
>hand, if there is a specific stoichiometry, there should be a corresponding
>density for the complex. Others have used sucrose gradient centrifugation
>(max % was 30%) and have manually collected fractions to analyze a similar
>complex.  We are wondering if the analytical ultracentrifuge can be used
>with a solute that is heavy enough to generate its own gradient.  We do not
>want to use CsCl because we want a constant and controllable ionic
>strength.  Metrizamide would seem to be heavy enough to form a gradient.
>Our question is whether it is a standard procedure in the analytical
>ultracentrifuge to do analyses with spin-formed gradients, and specifically
>whether metrizamide or some other non-ionic solute has been used.  We
>expect a density of the particle of about 1.12 and the size will vary from
>perhaps 100-1000 mu.  Obviously, we could do this with a regular centrifuge
>and fractionate the gradient.  It seemed to us that the precision and lack
>of extra steps would make the analytical instument a better choice.


Kind regards,

Kate
--
*****************************************************************
Dr.Katharina Spiegel		Manager, Keck Biophysics Facility
Off:	847-467-6533		Northwestern University
Lab:	847-467-6534		Department BMBCB
Fax:	847-467-6489		2153 N. Sheridan Road
Email: k-spiegel@nwu.edu	Evanston, IL 60208 USA

http://x.biochem.nwu.edu/Keck/keckmain.html

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

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Dear RASMBers:


I take care of a multi-user XLA and I was recently asked by an user
whether there is any information on  how to do a density gradient run
with the XLA. Hereby I would like to forward the more detailed
explanation of the experimental problem to this group of experts. Any
advice for us?



>	We would like to examine a lipid-DNA complex by centrifugation.  We
think

>that this complex will form at a particular stoichiometry with one or
the

>other of the components being in excess.  The lipid can be labelled
to

>aborb in a part of the spectrum different from DNA absorbance.  The

>particle sizes are quite variable, so velocity centrifugation will not
give

>us information on composition, although it may be useful eventually
for

>assessing the relationship between size and composition.  On the
other

>hand, if there is a specific stoichiometry, there should be a
corresponding

>density for the complex. Others have used sucrose gradient
centrifugation

>(max % was 30%) and have manually collected fractions to analyze a
similar

>complex.  <bold>We are wondering if the analytical ultracentrifuge can
be used

>with a solute that is heavy enough to generate its own
gradient.</bold>  We do not

>want to use CsCl because we want a constant and controllable ionic

>strength.  Metrizamide would seem to be heavy enough to form a
gradient.

><bold>Our question is whether it is a standard procedure in the
analytical

>ultracentrifuge to do analyses with spin-formed gradients, and
specifically

>whether metrizamide or some other non-ionic solute has been
used.</bold>  We

>expect a density of the particle of about 1.12 and the size will vary
from

>perhaps 100-1000 mu.  Obviously, we could do this with a regular
centrifuge

>and fractionate the gradient.  It seemed to us that the precision and
lack

>of extra steps would make the analytical instument a better choice.



Kind regards,


Kate 

-- 

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

Dr.Katharina Spiegel		Manager, Keck Biophysics Facility

Off:	847-467-6533		Northwestern University

Lab:	847-467-6534		Department BMBCB

Fax:	847-467-6489		2153 N. Sheridan Road

Email: k-spiegel@nwu.edu	Evanston, IL 60208 USA


http://x.biochem.nwu.edu/Keck/keckmain.html


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