Use and Applications of Subtractive Antibody Screening

Information about where and when genes are expressed is critical to understanding the function of the proteins that they encode in both health and disease. This concept is driving robust technology development in the field of functional genomics. Gene chips and other gene array systems permit the expression of thousands of genes to be surveyed simultaneously. A limitation of these approaches is that differential expression is detected at the level of RNA. Due to regulatory mechanisms that operate at the translational and post-translational levels, the absolute amounts of mRNA expression of a particular gene may not reflect the levels of its protein. Nevertheless, it is the level of protein that is ultimately more informative biologically in most cases. Another limitation is that gene expression data do not reveal important functional details, such as secondary modifications of proteins or their subcellular localization. The emerging field of proteomics addresses these limitations by working at the protein level to identify differential expression. By this approach, proteins in complex mixtures, such as cell lysates, are separated from one another chromatographically or electrophoretically, and then identified by such methods as microsequencing or mass spectroscopy, both of which are costly and labour intensive. We developed subtractive antibody screening (SAS) (Scherer etal., 1998) as a tool for functional proteomics (Figure 15.1). Our goal was to create a method by which particular subsets of differentially expressed proteins could be identified and their corresponding cDNAs cloned systematically. Further, we required that the method not depend on high cost equipment or services. In short, SAS relies on the generation