Figure 2.8 Western analysis. This figure follows the relative level of the cell-cycle-regulated protein Farlp during the course of about two cell divisions. Cells were synchronized in G1 (by depletion of an essential cyclin). The addition of galactose allows the cells to initiate a new round of cell division by restoring the expression of the cyclin. Panel (A) follows cell division by monitoring the level of unbudded cells (□) and binucleate cells (x), both determined by microscopic observation. Protein extracts were prepared from the cells in panel (A) and subjected to Western analysis using anti-Farlp antibody. The results of the Western analysis are shown in panel (B) and indicate that Farl protein accumulates in G1 prior to Start. Farlp runs as a close doublet of bands suggesting that the protein is found in a phosphorylated form. Taken from McKinney et al. (1993). Reproduced by permission of Cold Spring Habor Laboratory Press
Typically, the detection assay produces a colored, luminescent, or fluorescent precipitate on the surface of the membrane. This is then photographed, detected by X-ray film, or scanned by some form of densitometer for a permanent record and quantification where possible. Figure 2.8 illustrates the results of a Western analysis of Farlp during a cell division cycle (McKinney et al., 1993). The reader should note that often in addition to the level of the protein of interest the level of another protein, one considered to be relatively constant in amount during the cell cycle such as actin, is also determined using the same membrane. This protein serves as a control demonstrating that equal amounts of extract were loaded into each lane of the SDS-PAGE gel.
The different assay methods vary in their ability to be evaluated quantitatively. Moreover, determination of amounts is always relative to a standard sample run (for example the control condition) in the same gel. Some methods, such as chemi-luminescence, are linear over only a single log while others, such as those using a fluorescent secondary antibody, can be linear over several logs. Primary antibodies, secondary antibodies, and detection kits are commercially available and a wide variety of products for all aspects of Western analysis can be obtained from several suppliers.
For detailed information on Western analysis Ausubel et al. (2001) is an excellent starting point and provides updated procedures, theoretical discussions, and additional references. Others references listed below are also valuable resources. In addition, there is an abundant technical literature available from the commercial suppliers (Molecular Probes for example) evaluating the capabilities of the various products on the market as well as technical information from instrumentation manufacturers like Molecular Dynamics that manufactures densitometer, phos-phorimager, and fluorimeter equipment.
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