**5.3 Stoichiometry**

Determining the important parameter that is stoichiometry is not as easy a task as it seems. The apparent weight changes estimated from the complexes' gel mobility are not applicable in determining the stoichiometry due to complications of charges and conformational effects on gel migration. A different approach is needed. The presence of truncated or extended protein derived from the wild-type but with the same binding and multimerization capacity will originate new bands that can reflect the monomers bound to the nucleic acid (Hope & Struhl, 1987). A similar method that will be discussed in the next segment is the supershift EMSA that uses an antibody specific for the binding protein recognizing an epitope that is accessible while the protein is bound to the nucleic acid. The addition of the antibody to the preformed complex can provide an estimate of the number of proteins bound by the extent of increments in retardation (Michael N & Roizman B, 1991 as cited in Lane & Prentki, 1992).

A more complex approach has been proposed in 1988 to determine a complex's stoichiometry (Granger-Schnarr et al., 1988). After the separation of the free and the complexed nucleic acid on a non-denaturing gel, the proteins are transferred to a membrane after sodium dodecyl sulfate (SDS) denaturation. This then allows the detection of proteins directly or indirectly using a specific antibody. The protein bands as well as the nucleic acids autoradiograph are then quantified by densiometry and the relative stoichiometry can be determined. The need for a specific antibody limits this method to complexes formed by well known proteins with available antibodies.
