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8.  You measure the Kdiss of YFDB on specific and non-specific DNA using footprinting and you get 1 x 10-10 and 1x 10-6, respecitively.  Being a thorough biochemist, you repeat the experiment using using filter binding.  Using this approach, your specific Kdiss is 2 x 10-10, and your non-specific Kdiss is 5 x 10-4.  
a) Explain the essence of each method.  See lecture notes  
b) Why do you think the methods are more different for the not non-specific Kdiss than for the specific Kdiss?
c) Which result do you think is more reliable?

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b。Filter binding is a non-equilibrium method.  As soon as you bind the protein DNA complex to the filter, it will begin to dissociate, and it will continue to dissociate during the washes.  The magnitude of this effect will be particularly pronounced for the weak binding to non-specific DNA where your off-rate is high.  The interaction with specific DNA is much tighter.  Therefore, dissociation is not such a problem and the two techniques give essentially  the same result.
c。footprinting, since it’s an equilibrium method.

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9.  Estimate the molecular weight in Daltons, of the nucleosome core particle.

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This question requires you to know the composition of the particle and the approximate MWs of the proteins (partial credit for composition alone).
6 x 14, 000 D (H2A. H2B, H3)
2 x 10,000 D (H4)
1 x 146 x 660 = 96,000 (DNA)
Total = 200 kD

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