Density Gradient Centrifugation

Density Gradients & Making Them QUICKLY!
(density = mass/volume; equivalent term = specific gravity)

Density gradients are used in many different operations:

There are several ways for making density gradients including those that use syringes, twin linked containers, and other devices. Here are two very simple additional ways to make linear density gradients:

  1. This might be simple but it is one that takes many hours: fill a plastic centrifuge tube with - say - a 10% sucrose solution. Put it in the freezer. The first part that freezes will be almost pure water at the top, with only a little sucrose trapped in it, but as the overlying ice layer gets thicker, more and more sucrose is trapped within it. Thus when it is completely frozen, the top has little sucrose and the bottom has much. Upon subsequent thawing, the bottom melts first, and the melting proceeds upwards reinforcing the preparation of the gradient.

  2. Here is one that takes about 60 seconds of time from start to finish!

    The beauty of this procedure, which was invented by this author with the help of a remarkable undergraduate, is that it is not limited to centrifuge tubes. Cylinders of any size will work.

    1. Isopycnography (Equilibrium Density Gradient Centrifugation), which is used for separating things on the basis of differing intrinsic densities. This is a "static" system as the experimental objects come to rest at points in the tube at which they are in density equilibrium with the surrounding solvent at that point.

    2. Sedimentation Centrifugation, which is used to separate things on the basis of their differing "effective" sizes, or, roughly, on the basis of molecular weights. This is a "dynamic" situation because the experimental objects have started at the top and are settling through the gradient at rates roughly proportionate to the square roots of their molecular weights.

      In all instances, a centrifuge tube is filled with an aqueous solution that gradually increases in concentration of solute with depth. Depending on which of the two above uses is desired, different solutes are used.

      1. In isopycnography the solvent at the bottom of the tube must have a density greater than any of the experimental objects in the tube. Some favorite solutes are the various salts of expensive cesium (caesium) such as CsCl, Cs2SO4, or CsFormate, and sodium bromide (NaBr). Commonly employed gradients go from specfic gravities of 1.1 (top) to 1.8 (bottom). The centrifuges are usually run at speeds of over 30,000 rpm for days so that nucleic acids or proteins can migrate to their isopycnotic points where they then float at equilibrium. A great deal of the early work in molecular biology employed isopycnography until the discovery by Agnar Nygaard (visiting prof at the Univ. of Illinois from the University of Oslo) that nitrocellulose tightly bound many of the molecules.

      2. In sedimentation centrifugation, the solute is sucrose or some other not very dense substance that has appreciable viscosity. The experimental substances (usually particles) are overlayered atop the gradient, which is then centrifuged for up to 20 hours at about 20,000 rpm. It may be helpful to imagine this as Galileo's dropping the piece of lead shot and the lead cannon ball from the top of the Tower of Pisa through - not air - but molasses. The big cannon ball would hit the ground long before the bit of shot. In essence this is a matter of drag, which depends on the surface to mass ratio - the higher the ratio, the slower the descent. Thus, for example, the smaller part of a ribosome sinks much slower than the larger portion.

        For items that differ only in size and have the same density, Svedberg determined that the settling rate, S, had this relationship to molecular weight.

        S2/s2 = MW/mw

        Thus looking at the two major components of prokaryotic ribosomes, which are denominated as 16S and 23S, where "S" is the Svedberg unit, and can be thought of mnemonically as Settling or Sedimenting rates:

        232/162 = MW/mw = 529/256 = approx. 2

        Thus the larger portion is about twice the size of the smaller portion in prokaryotic ribosomal subunits.

        Shape plays a significant role also: imagine two sheets of paper. One is wadded into a tight ball and the other remains a flat sheet. Both are dropped from a high elevation. Which reaches the ground first? Both are the same in every way except for shape, which impinges on the relative effective surface area.

        WARNING! Again! This is a system in which ALL the experimental particles in the tube will eventually go to the bottom of the tube if the tube is centrifuged too long. (In isopycnography, on the other hand, which is an "equilibrium" system, one theoretically cannot centrifuge for too long.)

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