Analysis of Brain-Specific Gene Products

This chapter is concerned with the question, “Now that I’ve isolated a cDNA clone, what do I do with it?” The discussion is directed particularly at the analysis of clones of mRNAs encoding proteins of unknown identity that have been selected by procedures such as subtractive hybridization (see chapter by Travis et al. 59 ). In these cases, the question can be rephrased as, “What can I learn about the properties of the putative encoded protein that will give me clues as to its function?” Much can be gained from analyzing the sequence of the putative protein, determining the regional and cellular sites of expression of the protein and its mRNA, and their temporal expression during development. This type of analysis, which will also be of use to those studying clones of identified mRNAs, can often provide sufficient information to generate hypotheses for the function of the protein. We will also discuss procedures for generating antibodies against a protein encoded by a cDNA clone, using synthetic peptides or proteins generated in expression systems as immunogens. These antibodies can be used to demonstrate that the putative protein does indeed exist, to define its biochemical properties, and to test the functional hypotheses experimentally.