Gene Expression Analysis : Protein analysis

The kinds and amounts of proteins in cells do not always directly correspond to the amounts of mRNA present. Some mRNA are translated more efficiently than others, and some proteins undergo posttranslational modification. When analyzing the abundance and interactions of a large number of proteins, automated methods involving a variety of techniques, such as mass spectrometry and two-dimensional electrophoresis, are used.
When investigating one, or a limited number of proteins, labeled antibodies (Ab) are used to detect and quantify specific proteins and to determine posttranslational modifications.
1. Enzyme-linked immunosorbent assays: These assays (known as ELISA) are performed in the wells of a microtiter dish. The antigen (protein) is bound to the plastic of the dish. The probe used consists of an Ab specific for the protein (such as troponin) to be measured. The Ab is covalently bound to an enzyme, which will produce a colored product when exposed to its substrate. The amount of color produced is proportional to the amount of Ab present and, indirectly, to the amount of
protein in a test sample.
2. Western blots: Western blots (also called immunoblots) are similar to Southern blots, except that it is protein molecules in the sample that are separated by electrophoresis and blotted (transferred) to a membrane. The probe is a labeled Ab, which produces a band at the location of its antigen.
3. Detecting exposure to human immunodeficiency virus: ELISA and western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV Ab present in a patient’s blood sample. ELISA are used as the primary screening tool because they are very sensitive. Because these assays sometimes give false positives, however, western blots, which are more specific, are often used as a confirmatory test (Fig. 1). [Note: ELISA and western blots can only detect HIV exposure after anti-HIV Ab appear in the bloodstream. PCRbased testing for HIV is more useful in the first few months after exposure.]

Figure 1: Testing for human immunodeficiency virus (HIV) exposure by enzyme-linked immunosorbent assays (ELISA) and western blots.