Immunoprecipitation is a useful variation on the theme of affinity chromatography. Specific antibodies that recognize the protein to be purified are attached to small agarose beads. Rather than being packed into a column, as in affinity chromatography, a small quantity of the antibody-coated beads is simply added to a protein extract in a test tube and mixed in suspension for a short period of time— thereby allowing the antibodies to bind the desired protein. The beads are then collected by low-speed centrifugation, and the unbound proteins in the supernatant are discarded. This method is commonly used to purify small amounts of enzymes from cell extracts for analysis of enzymatic activity or for studies of associated proteins.
bead with covalently attached substrate
Figure 8–9 Three types of matrices used for chromatography. (A) In ion-exchange chromatography, the insoluble matrix carries ionic charges that retard the movement of molecules of opposite charge. Matrices used for separating proteins include diethylaminoethylcellulose (DEAE-cellulose), which is positively charged, and carboxymethylcellulose (CM-cellulose) and phosphocellulose, which are negatively charged. Analogous matrices based on agarose or other polymers are also frequently used. The strength of the association between the dissolved molecules and the ion-exchange matrix depends on both the ionic strength and the pH of the solution that is passing down the column, which may therefore be varied systematically (as in Figure 8–10) to achieve an effective separation. (B) In gel-filtration chromatography, the small beads that form the matrix are inert but porous. Molecules that are small enough to penetrate into the matrix beads are thereby delayed and travel more slowly through the column than larger molecules that cannot penetrate. Beads of cross-linked polysaccharide (dextran, agarose, or acrylamide) are available commercially in a wide range of pore sizes, making them suitable for the fractionation of molecules of various molecular mass, from less than  500 daltons to more than 5 × 106 daltons.
(C) Affinity chromatography uses an insoluble matrix that is covalently linked to a specific ligand, such as an antibody molecule or an enzyme substrate, that will bind a specific protein. Enzyme molecules that bind to immobilized substrates on such columns can be eluted with a concentrated solution of the free form of the substrate molecule, while molecules that bind to immobilized antibodies can be eluted by dissociating the antibody–antigen complex with concentrated salt solutions or solutions of high or low pH. High degrees of purification can be achieved in a single pass through an affinity column.
pool these fractions and apply them to the next column below fraction number pool these fractions and apply them to the next column below pool these fractions, which now contain the highly purifed protein
Genetically Engineered Tags Provide an Easy Way to Purify Proteins
Using the recombinant DNA methods discussed in subsequent sections, any gene can be modified to produce its protein with a special recognition tag attached to it, so as to make subsequent purification of the protein simple and rapid. Often the recognition tag is itself an antigenic determinant, or epitope, which can be recognized by a highly specific antibody. The antibody can then be used to purify the protein by affinity chromatography or immunoprecipitation (Figure 8–11). Other types of tags are specifically designed for protein purification. For example, a repeated sequence of the amino acid histidine binds to certain metal ions, including nickel and copper. If genetic engineering techniques are used to attach a short string of histidines to one end of a protein, the slightly modified protein can be retained selectively on an affinity column containing immobilized nickel ions. Metal affinity chromatography can thereby be used to purify the modified protein from a complex molecular mixture.