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Laura and all who may be interested,<br>
<br>
I presume that you would like to perform equilibrium sedimentation in a
density gradient (buoyant density centrifugation) and not band or
analytical zone centrifugation for velocity characterization. You
message indicated that you would liike to study protein-DNA interactions.
I envision that you have a large DNA molecule that you wish to study
protein binding since the power of buoyant density centrifugation is the
detection of buoyant density shifts due to binding of a ligand or
protein. Since the width of the band formed at equilibrium is inversely
proportional to Mw large DNA molecules form sharp bands and ligand
binding will shift the band position and this is readily
measureable.<br>
The buoyant density shift will depend on the change in hydrated
buoyant density caused by the number of ligands bound. Bauer and Vinograd
developed the theory for evaluating the binding of ethidium bromide to
DNA. We applied the theory to another problem and the reference is given
below:<br>
<font face="Arial, Helvetica">Beerman, T.A. and J. Lebowitz.
1973. Further analysis of the altered secondary structure of
superhelical DNA. Sensitivity to methylmercuric hydroxide, a
chemical probe for unpaired bases. J. Mol. Biol.,79:451-470.<br>
This reference should have sufficient detail for setting up experiments.
However, we used CsSO4 since Cl ions would out compete DNA for CH3Hg
binding. The most detailed reference to the methodology is the review by
Vinograd and Hearst 1962, </font>Equilibrium Sedimentation of
Macromolecules in a Density Gradient, Fortschritte der Chemie organischer
Naturstoff (Progress in the Chemistry of Organic Natural Products) Vol.
XX Wien, Springer-Verlag. There are practical details in this
review on p.395-7 with citations. Also<br>
Check out other papers by Hearst, John.<br>
<br>
Although, protein-nucleic acid interactions can be stable in CsCl
gradients, i.e. viruses and ribosomes I would suspect that many <br>
protein-DNA interactions would be disrupted due the breaking of
electrostatic interactions at high concentrations of CsCl. <br>
The practical details for making CsCl solutions are not difficult. Tables
are available of the density of CsCl vs. refractive index. <br>
Based on these measurements the equation that you need to use is
density<br>
p=10.8601xn (ref.index) -13.4974<br>
Hence if you have a refractometer you can readily measure the density of
the solution. <br>
The most difficult aspect of the experiment is insure that the density of
the protein DNA complex will band in the range of the density gradient
that will be formed. I strongly suggest that you band DNA first to become
familiar with the methodology. It is also a very good idea to use a
marker DNA with a known but very different buoyant density. We use to be
able to buy these bacterial DNAs but I don't know if they are available
today.<br>
<x-tab> </x-tab> I hope
that this terse email will be a sufficient introduction to get you
started. You will need to <br>
read the old literature to get some of the theoretical concepts. Van
Holde et al. Biophysical Chemistry text covers the essential theory. If
you can get high Mw DNA to band and determine the buoyant density you
will be on your way.<br>
<br>
At 05:17 PM 10/2/02 +0200, Laura.Giangiacomo@uniroma1.it wrote:<br>
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<br>
Dear all,<br>
next week I will start to do some experiments about protein-DNA
interaction<br>
in a CsCl gradient. As I never worked with CsCl gradients, I need to
know<br>
almost everything, e.g. how to prepare a CsCl saturated solution, how
to<br>
handle the sample and how to analyze the data (I guess there should be
a<br>
density distribution of CsCl and hence of the different
macromolecular<br>
species as a function of radius and rotor speed). I must add that I do
not<br>
have a densitymeter, so I can't make direct density measurements.<br>
Is there any review available or is there anybody who works on these
topic<br>
and who can help me?<br>
Thank you very much in advance.<br>
<br>
Sincerely,<br>
<br>
Laura Giangiacomo<br>
<br>
----<br>
Dr. Laura Giangiacomo<br>
Dept. of Biochemical Sciences<br>
University of Rome "La Sapienza"<br>
P.le Aldo Moro 5 - 00185 Roma (Italy)<br>
Phone +39 (0)6 49910990<br>
Fax +39 (0)6 4440062<br>
<br>
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<font face="Times New Roman, Times">Jacob Lebowitz, PhD <br>
Molecular Interactions Resource <br>
Division of Bioengineering and Physical Science, ORS <br>
National Institutes of Health <br>
Mail: Bldg. 13 Rm. 3N17<br>
Office Bldg. 13 Rm. 3E49<br>
13 South Drive <br>
Bethesda, MD 20892 - 5766 <br>
Tel: (301) 435-1955 <br>
Fax: (301) 480-1242<br>
</font>email: lebowitz@helix.nih.gov<br>
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