[RASMB] CsCl gradient

[Jacob Lebowitz]

Laura and all who may be interested,

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.
   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:
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.
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, 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
Check out other papers by Hearst, John.

Although, protein-nucleic acid interactions can be stable in CsCl 
gradients, i.e. viruses and ribosomes I would suspect that many
protein-DNA interactions would be disrupted due the breaking of 
electrostatic interactions at high concentrations of CsCl.
The practical details for making CsCl solutions are not difficult. Tables 
are available of the density of CsCl vs. refractive index.
Based on these measurements the equation that you need to use is density
p=10.8601xn (ref.index) -13.4974
Hence if you have a refractometer you can readily measure the density of 
the solution.
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.
          I hope that this terse email will be a sufficient introduction to 
get you started. You will need to
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.

At 05:17 PM 10/2/02 +0200, Laura.Giangiacomo at uniroma1.it wrote:
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>Dear all,
>next week I will start to do some experiments about protein-DNA interaction
>in a CsCl gradient. As I never worked with CsCl gradients, I need to know
>almost everything, e.g. how to prepare a CsCl saturated solution, how to
>handle the sample and how to analyze the data (I guess there should be a
>density distribution of CsCl and hence of the different macromolecular
>species as a function of radius and rotor speed). I must add that I do not
>have a densitymeter, so I can't make direct density measurements.
>Is there any review available or is there anybody who works on these topic
>and who can help me?
>Thank you very much in advance.
>
>Sincerely,
>
>Laura Giangiacomo
>
>----
>Dr. Laura Giangiacomo
>Dept. of Biochemical Sciences
>University of Rome "La Sapienza"
>P.le Aldo Moro 5 - 00185 Roma (Italy)
>Phone +39 (0)6 49910990
>Fax +39 (0)6 4440062
>
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Jacob Lebowitz, PhD
Molecular Interactions Resource
Division of Bioengineering and Physical Science, ORS
National Institutes of Health
Mail: Bldg. 13 Rm. 3N17
Office Bldg. 13 Rm. 3E49
13 South Drive
Bethesda, MD 20892 - 5766
Tel: (301) 435-1955
Fax: (301) 480-1242
email: lebowitz at helix.nih.gov
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