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From the literature I read, I came to understand the following. Please correct me if wrong.

  1. In CsCl gradient centrifugation, the gradient is achieved by centrifuging the CsCl solution at high rpm, which causes the heavy Cs ions to distribute unevenly throughout the solution, due to sheer centrifugal force.

  2. In Sucrose gradient, solutions of decreasing concentrations of sucrose are layered one over the other slowly.

Why can't we achieve the sucrose gradient by simply centrifuging it like in the CsCl gradient?

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Sucrose gradient separations are an example of a rate-zonal centrifugation technique (fair technical document on centirfugation separations). The idea is you layer lighter sucrose solutions on top of one another, for example, 35% at the bottom of the tube (the most dense zone) and 15% at the top band (the least dense zone). There might be 5 or 6 layers. You then layer the sample, which may contain cell lysate, at the very top.

This method is done on a centrifuge, and the idea is that in order to penetrate down to a more dense layer, the component of the lysate must exceed the density of the preceeding sucrose layer. Quite literally the dense components are pushing down through the sucrose layers. The method is suitable for organelles and proteins. The layering must be done deliberately and carefully, however, because the sucrose layers and sample will easily mix if they're disturbed. The same concept applies to ficol density gradient separations: If there's no density of solution below the sample for it to pass through, there's no separation.

On the other hand, the CsCl method is an example of an isopycnic centrifugation suitable for the separation of nucleic acids, see the above mentioned reference. This is because the DNA migrates to where the denisty of the DNA equals the density of the gradient, referred to as the neutral buoyancy or isopycnic point.

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    $\begingroup$ Thanks for the answer. But, if you have a solution of sucrose of a particular concentration and you centrifuge it at high rpm, would it be enough to form the density gradient like the CsCl, or is it necessary to layer solutions of varied concentrations and then centrifuge? Why exactly do we need solutions of different concentration unlike CsCl method where we use just one solution of CsCl of a specific concentration? $\endgroup$
    – Arjun Devdas
    Commented Mar 4, 2018 at 15:55
  • $\begingroup$ It's necessary to layer the sample on top of the sucrose solution. In the CsCl method the sample is in suspension and works on a different principle. If you suspend your sample in the sucrose solution, you won't accomplish anything. For example, if you want really dense things like organelles, you could put a single dense layer of sucrose under the sample and spin that, but you'd only obtain one fraction: The constituents with the highest density. $\endgroup$
    – CKM
    Commented Mar 4, 2018 at 18:29
  • $\begingroup$ The principle behind the sucrose gradient is denser things push down from layer to layer until they're no longer dense enough to get through and end up as a band somewhere along the gradient, or pellet completely to the bottom. $\endgroup$
    – CKM
    Commented Mar 4, 2018 at 18:31
  • $\begingroup$ So, if you mix them together and then spin, everything will just pellet to the bottom and you get no separation. $\endgroup$
    – CKM
    Commented Mar 4, 2018 at 18:32

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