chapter  7
30 Pages

Analysis of the proteome

As discussed in the previous chapter, the only true measure of gene expression is a measure of protein production. In some situations, posttranscriptional control events mean that changes in protein level do not run parallel with changes in transcript level (this is more commonly the case in eukaryotes than in prokaryotes). It would be surprising to find situations where the transcript level for a gene increased whilst the protein level decreased, and the reverse is even less likely, but to find no change, or more usually a disproportionate change in protein levels compared with transcript levels is common. Thus, interpreting the results of experiments that have determined transcript levels can be difficult, and can sometimes lead to incorrect conclusions concerning changes in gene expression. To give an accurate picture of gene expression, therefore, and if you do not want to use reporter gene approaches as described in Chapter 6, you will have to measure protein levels directly. If all you want to do is confirm transcriptomics experiments, and have therefore identified a small number of test proteins whose abundances you want to measure in a pair of cell extracts, one relative to the other, then a number of different possible methods exist that will allow you to do this. However, all these methods require some degree of prior knowledge concerning each test protein. You either need to have an antibody that recognizes the protein (Section 6.3 and below) or you need to know its ligand binding properties (the focus of this section) or have an assay for its enzyme activity if any (see Section 6.2). If none of this prior knowledge applies, then you might have to use proteomics to separate all the proteins in a cell extract, locate the spot representing the test protein, and then quantify the intensity of this protein spot when different cell extracts, each made from cells growing in a different physiological state, are subjected to the same separation protocols.