**Part 3**

**Electrophoresis Application in the Analysis of Protein-Nucleic Acid Interaction and Chromosomal Replication** 

202 Gel Electrophoresis – Advanced Techniques

Singh A, Goering RV, Simjee S, Foley SL, Zervos MJ. 2006. Application of molecular techniques to the study of hospital infection. Clin Microbiol Reviews; 19 (3) 512 -3 Tenover FC, Abreit RD, Goering RV, Michelsen PA, Murray BE, Persing DH, et al. 1995.

Woodford N, Fagan EJ, Ellington MJ. 2006. Multiplex PCR for rapid detection of genes

*Enterococcus faecalis*. J. Clin. Microbiol. 35:969–972.

57(1):154-5

Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol; 33:2233—9. Thal, L. A., J. Silverman, S. Donabedian, and M. J. Zervos. 1997. The effect of Tn*916* 

insertions on contour-clamped homogenous electrophoresis patterns of the

encoding CTX-M extended-spectrum B-lactamases. J Antimicrobial Chemotherapy;

**12** 

*Portugal* 

**Electrophoretic Mobility Shift Assay:** 

*Institute of Hygiene and Tropical Medicine, New University of Lisbon* 

Carolina Alves and Celso Cunha *Center for Malaria and Tropical Diseases* 

**Analyzing Protein – Nucleic Acid Interactions** 

Interactions between proteins and nucleic acids mediate a wide range of processes within a cell from its cycle to the maintenance of cellular metabolic and physiological balance. These specific interactions are crucial for control of DNA replication and DNA damage repair, regulation of transcription, RNA processing and maturation, nuclear transport, and

The characterization of protein-nucleic acid interactions is essential not only for understanding the wide range of cellular processes they are involved in, but also the mechanisms underlying numerous diseases associated with the breakdown of regulatory systems. These include, but are far from being limited to, cell cycle disorders such as cancer and those caused by pathogenic agents that rely on or interfere with host cell machinery. More recently, it has been hypothesized that many neurological disorders such as Alzheimer's, Huntington's, Parkinson's, and polyglutamine tract expansion diseases are a consequence, at least in part, of aberrant protein-DNA interactions that may alter normal

The electrophoretic mobility shift assay (EMSA), also known as gel retardation assay, is a regularly used system to detect protein-nucleic acid interactions. It was originally developed with the aim of quantifying interactions between DNA and proteins (Fried & Crothers, 1981; Garner & Revzin, 1981) and since then evolved to be suitable for different purposes including the detection and quantification of RNA-protein interactions. EMSA is most commonly used for qualitative assays including identification of nucleic acid-binding proteins and of the respective consensus DNA or RNA sequences. Under proper conditions, however, EMSA can also be used for quantitative purposes including the determination of

EMSA is a commonly used method in the characterization of transcription factors, the most intensely studied DNA-binding proteins, and the largest group of proteins in humans, second only to metabolic enzymes. Their purification and identification is crucial in understanding gene regulatory mechanisms. Transcription factors are sequence specific DNA binding proteins that are usually assembled in complexes formed prior to transcription initiation. They bind discreet and specific DNA sequences in the promoter

**1. Introduction**

translation.

patterns of gene expression (Jiménez, 2010).

binding affinities, kinetics, and stoichiometry.
