**4. The method**

There are five focal steps in a conventional EMSA protocol that involve different variables susceptible to optimization: (1) preparation of protein sample; (2) synthesis and labeling of nucleic acid; (3) binding reaction; (4) non-denaturing gel electrophoresis and (5) detection of the outcome. In this segment we will discuss each step separately mentioning the key variables in each one and the options available for any given situation. Figure 2 represents schematically the regular steps in a gel retardation assay that will be discussed below. Whenever possible we will also refer to examples in the literature.

Fig. 2. A schematic representation of a conventional EMSA protocol. The labeled nucleic acid, simplified as lines with a star representing the label, is mixed with the protein sample, represented by the oval shapes, in a binding reaction and then loaded into a non-denaturing gel. After electrophoresis the result is detected according to the label in the nucleic acid. On the schematic gel (A) represents a well on which only the labeled nucleic acid was loaded. The free nucleic acid is expected to have more mobility than the bound molecules. In well (B) is symbolized a labeled nucleic acid binding to one small peptide and in well (C) is binding to two larger proteins. The heavier complex (in C) is expected to display the lowest mobility during electrophoresis and therefore is closer to the beginning of the gel.
