Purifying, Detecting, and Characterizing Proteins
■ Proteins can be separated from other cell components and from one another on the basis of differences in their physical and chemical properties.
■ Centrifugation separates proteins on the basis of their rates of sedimentation, which are influenced by their masses and shapes.
■ Gel electrophoresis separates proteins on the basis of their rates of movement in an applied electric field. SDS-polyacrylamide gel electrophoresis can resolve polypeptide chains differing in molecular weight by 10 percent or less (see Figure 3-32).
■ Liquid chromatography separates proteins on the basis of their rates of movement through a column packed with spherical beads. Proteins differing in mass are resolved on gel filtration columns; those differing in charge, on ionexchange columns; and those differing in ligand-binding properties, on affinity columns (see Figure 3-34).
■ Various assays are used to detect and quantify proteins. Some assays use a light-producing reaction or radioactivity to generate a signal. Other assays produce an amplified colored signal with enzymes and chromogenic substrates.
■ Antibodies are powerful reagents used to detect, quantify, and isolate proteins. They are used in affinity chro-matography and combined with gel electrophoresis in
Western blotting, a powerful method for separating and detecting a protein in a mixture (see Figure 3-35).
■ Autoradiography is a semiquantitative technique for detecting radioactively labeled molecules in cells, tissues, or electrophoretic gels.
■ Pulse-chase labeling can determine the intracellular fate of proteins and other metabolites (see Figure 3-36).
■ Three-dimensional structures of proteins are obtained by x-ray crystallography, cryoelectron microscopy, and NMR spectroscopy. X-ray crystallography provides the most detailed structures but requires protein crystallization. Cryo-electron microscopy is most useful for large protein complexes, which are difficult to crystallize. only relatively small proteins are amenable to NMR analysis.
teins, especially in the early stages in the formation of insoluble filaments.
Understanding the operation of protein machines will require the measurement of many new characteristics of proteins. For example, because many machines do nonchemical work of some type, biologists will have to identify the energy sources (mechanical, electrical, or thermal) and measure the amounts of energy to determine the limits of a particular machine. Because most activities of machines include movement of one type or another, the force powering the movement and its relation to biological activity can be a source of insight into how force generation is coupled to chemistry. Improved tools such as optical traps and atomic force microscopes will enable detailed studies of the forces and chemistry pertinent to the operation of individual protein machines.
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