3.5 Purifying, Detecting, and Characterizing Proteins

A protein often must be purified before its structure and the mechanism of its action can be studied in detail. However, because proteins vary in size, shape, oligomerization state, charge, and water solubility, no single method can be used to isolate all proteins. To isolate one particular protein from the estimated 10,000 different proteins in a particular type of cell is a daunting task that requires methods both for separating proteins and for detecting the presence of specific proteins.

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Any molecule, whether protein, carbohydrate, or nucleic acid, can be separated, or resolved, from other molecules on the basis of their differences in one or more physical or chemical characteristics. The larger and more numerous the differences between two proteins, the easier and more efficient their separation. As a practical matter, the more abundant a particular protein is in a biological sample, the easier it is to separate it from the other molecules in the sample. The three most widely used characteristics for separating proteins are size, defined as either length or mass; net electrical charge; and affinity for specific ligands. In this section, we briefly outline several important techniques for separating proteins; these separation techniques are also useful for the separation of nucleic acids and other biomolecules. (Specialized methods for removing membrane proteins from membranes are described in Chapter 7 after the unique properties of these proteins are discussed.) We then consider the use of radioactive compounds for tracking biological activity. Finally, we consider several techniques for characterizing a protein’s mass, sequence, and three-dimensional structure.