Key Concepts of Section 3.5

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 (see Figure 3-37).

• Electrophoresis separates proteins on the basis of their rates of movement in an applied electric field. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) can resolve polypeptide chains differing in molecular weight by 10 percent or less (see Figure 3-38). Two-dimensional gel electrophoresis provides additional resolution by separating proteins first by charge (first dimension) and then by mass (second dimension).

• 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 ion-exchange columns; and those differing in ligand-binding properties, on affinity columns (see Figure 3-40).

• Various assays are used to detect and quantify proteins. Some assays use a light-producing reaction to generate a readily detected signal. Other assays produce an amplified colored signal with enzymes and chromogenic substrates.

• Antibodies are powerful reagents used to detect, quantify, and isolate proteins.

• Immunoblotting, also called Western blotting, is a frequently used method to study specific proteins that exploits the high specificity and sensitivity of protein detection by antibodies and the high-resolution separation of proteins by SDS-PAGE (see Figure 3-41).

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• Immunoprecipitation, often abbreviated as IP, permits the separation of a protein of interest from other proteins in a complex mixture using antibodies specific for the protein of interest. The antibodies are used to precipitate their target protein out of solution for subsequent analysis. Molecules tightly bound to the target protein can precipitate with it (co-immunoprecipitation).

• Isotopes, both radioactive and nonradioactive, play a key role in the study of proteins and other biomolecules. They can be incorporated into molecules without changing the chemical composition of the molecule or as add-on tags. They can be used to help detect the synthesis, location, processing, and stability of proteins.

• Autoradiography is a technique for detecting radioactively labeled molecules in cells, tissues, or electrophoretic gels using two-dimensional detectors (photographic emulsion or electronic detectors).

• Pulse-chase experiments can determine the intracellular fate of proteins and other metabolites (see Figure 3-42).

• Mass spectrometry is a very sensitive and highly precise method of detecting, identifying, and characterizing proteins and peptides.

• 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. Cryoelectron microscopy is most useful for large protein complexes, which are difficult to crystallize. Only relatively small proteins are amenable to NMR three-dimensional structural analysis.