Figure 1. Sucrose density gradient centrifugation. The sample containing ribosomes, ribosomal subunits or polysomes is layered on top of a buffer solution in which the concentration of sucrose increases from top to bottom in a centrifuge tube. The tubes are then spun in an ultracentrifuge in a horizontal position so that the centrifugal force is applied along the longitudinal axis of the tube. The heavier the particles, the farther they will move into the gradient. The centrifugation time depends on the types of particles to be separated. It is shortest for polysomes and longest for ribosomal subunits. After centrifugation, the gradient is fractionated and the A₂₆₀ of the fractions is monitored.

Figure 2. Typical A₂₆₀ profiles obtained after sucrose density gradient centrifugation of (a) intact ribosomes, (b) ribosomal subunits, where the first peak corresponds to the small subunit, the second one to the large subunit, and (c) polysomes, where the first peak corresponds to monosomes (i.e. one ribosome attached to mRNA) and subsequent peaks to mRNA carrying two, three, etc. ribosomes. Note that the centrifugation time is different in each case (shortest for polysomes, longest for ribosomal subunits). The high absorbancy at the top of the gradient (left) is due to tRNA and free protein.

Figure 3. Tandem affinity purification (TAP) of pre-ribosomal particles. A cell extract is prepared from cells that have been genetically altered to express a specific, nonribosomal protein that is part of the pre-ribosomal particle to be purified, linked to a ‘tag’. The latter consists of an epitope of Staphylococcus aureus protein A connected to the calmodulin-binding peptide (CBP) via a cleavable linker peptide containing the consensus recognition site for the protease from tobacco etch virus (TEV). The extract is chromatographed on a column containing IgG-Sepharose beads to bind the tagged particles. After washing, the particles are released by cleaving the linker peptide with the TEV protease. This exposes the CBP, allowing the tagged particles to bind to a calmodulin–Sepharose column in the presence of  Ca²⁺ ions. Purification is completed by further washing and elution of the bound pre-ribosomes using a buffer containing EGTA, which removes the  Ca²⁺ ions required for binding to the column material.