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Typical results obtained when three different chromatographic steps are used in succession to purify a protein. In this example a homogenate of cells was first fractionated by allowing it to percolate through an ion-exchange resin packed into a column (A). The column was washed, and the bound proteins were then eluted by passing a solution containing a gradually increasing concentration of salt onto the top of the column. Proteins with the lowest affinity for the ion-exchange resin passed directly through the column and were collected in the earliest fractions eluted from the bottom of the column. The remaining proteins were eluted in sequence according to their affinity for the resin—those proteins binding most tightly to the resin requiring the highest concentration of salt to remove them. The protein of interest was eluted in several fractions and was detected by its enzymatic activity. The fractions with activity were pooled and then applied to a second, gel-filtration column (B). The elution position of the still-impure protein was again determined by its enzymatic activity and the active fractions were pooled and purified to homogeneity on an affinity column (C) that contained an immobilized substrate of the enzyme. (D) Affinity purification of cyclin-binding proteins from S. cerevisiae, as analyzed by SDS polyacrylamide-gel electrophoresis (see Figure 8-14). Lane 1 is a total cell extract; lane 2 shows the proteins eluted from an affinity column containing cyclin B2; lane 3 shows one major protein eluted from a cyclin B3 affinity column. Proteins in lanes 2 and 3 were eluted with salt and the gels was stained with Coomassie blue. (D, from D. Kellogg et al., J. Cell Biol. 130:675–685, 1995. © The Rockefeller University Press.)