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(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

For further informations, watch this video  and read this https://www.thermofisher.com/it/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/gel-shift-assays-emsa.html

https://www.thermofisher.com/it/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/gel-shift-assays-emsa.html