with another given protein. Cell extracts containing the presumed interaction complex are first immunoprecipitated with antibody against one of the proteins. The material isolated by this means is then tested for the presence of the other protein by immunoblotting with a specific antibody against the second protein.
A-14 Immunoblotting (Western blotting).
Like immunoprecipitation (see Section A-13), immunoblotting is used for identifying the presence of a given protein in a cell lysate, but it avoids the problem of having to label large quantities of cells with radioisotopes. Unlabeled cells are placed in detergent to solubilize all cell proteins, and the lysate is run on SDS-PAGE to separate the proteins (see Section A-13). The size-separated proteins are then transferred from the gel to a stable support such as a nitrocellulose membrane. Specific proteins are detected by treatment with antibodies able to react with SDS-solubilized proteins (mainly those that react with denatured sequences); the bound antibodies are revealed by anti-immunoglobulin antibodies labeled with an enzyme. The term Western blotting as a synonym for immunoblotting arose because the comparable technique for detecting specific DNA sequences is known as Southern blotting, after Edwin Southern, who devised it, which in turn provoked the name ‘Northern’ for blots of size-separated RNA, and ‘Western’ for blots of size-separated proteins. Western blots have many applications in basic research and clinical diagnosis. They are often used to test sera for the presence of antibodies against specific proteins, for example, to detect antibodies against different constituents of HIV (Fig. A.15).
A-15 Use of antibodies in the isolation and characterization of multiprotein complexes by mass spectrometry.
Many of the proteins that function in immune cells are components of multi-protein complexes. This is the case for cell-surface receptors, such as the T-cell and B-cell antigen receptors and most cytokine receptors, as well as intracellular proteins involved in signal transduction, gene expression, and cell death. Antibodies that bind to one member of such a complex can be used to identify the other members of the complex by a process of co-immunoprecipitation followed by Western blotting or mass spectrometry.
A mass spectrometer can provide extremely precise measurements of the masses of constituents in a preparation of molecules. To identify unknown proteins in a sample, such as that acquired by co-immunoprecipitation, the sample often is first subjected to one-dimensional SDS-PAGE or two-dimensional gel electrophoresis (see Section A-13) to separate the proteins in the complex for individual analysis. Gel slices are excised and treated with a proteolytic enzyme, such as trypsin, to digest the protein into a series of peptides that can be easily eluted from the gel. The peptide mixture is then introduced into the mass spectrometer, which ionizes the peptides, transfers them to the gas phase, and then separates them under high vacuum by subjecting them to a magnetic field. The separation is based on the mass/charge (m/z)
Fig. A.15 Western blotting is used to identify antibodies against the human immunodeficiency virus (HIV) in serum from infected individuals. The virus is dissociated into its constituent proteins by treatment with the detergent SDS, and its proteins are separated using SDS-PAGE. The separated proteins are transferred to a nitrocellulose sheet and reacted with the test serum. Anti-HIV antibodies in the serum bind to the various HIV proteins and are detected with enzyme-linked anti-human immunoglobulin, which deposits colored material from a colorless substrate. This general methodology will detect any combination of antibody and antigen and is used widely, although the denaturing effect of SDS means that the technique works most reliably with antibodies that recognize the antigen when it is denatured.