EXPERIMENTAL FIGURE 3-40 Three commonly used liquid chromatographic techniques separate proteins on the basis of mass, charge, or affinity for a specific binding partner. (a) Gel filtration chromatography separates proteins that differ in size. A mixture of proteins is carefully placed, or loaded, on the top of a cylinder packed with porous beads. Smaller proteins travel through the column more slowly than larger proteins. Thus the different proteins emerging in the eluate flowing out of the bottom of the column at different times (different elution volumes) can be collected in separate tubes, called fractions. (b) Ion-exchange chromatography separates proteins that differ in net charge in columns packed with beads that carry either a positive charge (shown here) or a negative charge. Proteins having the same net charge as the beads are repelled and flow through the column, whereas proteins having the opposite charge bind to the beads more or less tightly, depending on their structures. Bound proteins—in this case, negatively charged proteins—are subsequently eluted by passing a salt gradient (usually of NaCl or KCl) through the column. As the ions bind to the beads, they displace the proteins; more tightly bound proteins require higher salt concentrations in order to be released. (c) In antibody-affinity chromatography, a mixture of proteins is passed through a column packed with beads to which a specific antibody is covalently attached. Only proteins with high affinity for the antibody are retained by the column; all the nonbinding proteins flow through. After the column is washed, the bound protein is eluted with an acidic solution or some other solution that disrupts the antigen-antibody complexes; the released protein then flows out of the column and is collected.