Sucrose-Gradient Technique for determination of sedimentation coefficients (S)

    (a) A sucrose gradient, with continuously variable concentration of sucrose, is prepared in a centrifuge tube. The technique is similar to that used in making Irish Coffee or a Mexican Flag cocktail. (b) A sample of macromolecules is layered over the pre-formed sucrose gradient. (c) The tube is placed in an ultracentrifuge and spun at extremely high speed for several hours; the sucrose gradient remains stable. (d) Depending on the sizes & molecular weights of the macromolecular components, they migrate ("sediment") through the gradient at different rates: lighter molecules will move less quickly than more dense, compact molecules. Each molecular type will eventually form a discrete band at its isopycnic point, where its density equals that of the sucrose gradient. (e) The fractions can be recovered by poking a hole in the bottom of the centrifuge tube, and collecting a series of samples as a predetermined number of drops. The sample tubes are numbered in order from lighter to heavier.

    In early experiments with rRNA molecules, the various components were described according to the number of the sample tube in which they appeared. Small rRNA subunits appeared in fraction 5, and were designated as 5S , while large rRNA subunits appeared in fractions 16 & 23, and were designated 16S or 23S. These were subsequently designated as Svedberg units (S).

    NB: Gradient techniques are often misrepresented in introductory texts. An ultracentrifugal separation differs from the setup shown here by the use of special plastic tubes set in metal carriers. The carriers are hooked onto the underside of the ultracentrifuge head. At low speed, the tube swing up horizontal to the head and lock into place. Centrifugal forces may exceed 100,000 x g.

    Density gradient techniques can be used to separate other types of molecules, for example DNA molecules that vary in [G+C] content, and may employ other gradient substances, such as cesium chloride.