Back to index

(Karen Odin)

EMSA (electrophoretic mobility shift assay) or gel shift: technique used for studying gene regulation and determining protein–DNA interactions. EMSA can be used qualitatively to identify sequence-specific, DNA-binding proteins (such as transcription factors) in crude lysates and, in conjunction with mutagenesis, to identify the important binding sequences within the upstream regulatory region of a given gene. It can also be used quantitatively to measure thermodynamic and kinetic parameters.

Advantages:

• simple to perform
• robust enough to accommodate a wide range of binding conditions
• highly sensitive, allowing assays to be performed with small protein and nucleic acid concentrations and small sample volumes
• a wide range of nucleic acid sizes and structures are compatible with the assay
• the distribution of proteins between several nucleic acid molecules can be monitored within a single solution

Disadvantages:

• samples are not at chemical equilibrium during the electrophoresis step
• the electrophoretic mobility of a protein-nucleic acid complex depends on many factors other than the size of the protein
• the electrophoretic mobility of a complex provides little direct information about the location of the nucleic acid sequences that are occupied by protein
• the time resolution of the current assay is defined by the interval required for manual solution handling

EMSA uses native PolyAcrylamide Gel Electrophoresis (PAGE) to resolve a mixture of a protein of interest and a labeled DNA probe containing potential target sites of the protein. A DNA probe bound with protein will migrate slower compared with a free DNA probe and is therefore retarded in its migration through a polyacrylamide matrix. Radiolabeling of DNA by 32P has been the predominant method for detection in EMSAs.

Protocol:

1. Gel preparation
2. Preparation of infrared fluorescent dye-labeled probes
3. Preparation of unlabeled/cold probes (competitors)
4. Binding reaction and electrophoresis
5. Imaging