Student Wiki on methodology

This Wiki is intended to collectively make the point on methodologies employed in research papers we analyze during the course. "Writers" are students who wish to contribute to a specific subject. Before contributing, please add your name in the "Writers group choice". When initiating a contribution, please indicate your name in brackets.


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This page contains the links to the nine official subjects, which are the same in the Choice.

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Please do not make extensive cut-and-paste: it s useless, anybody can go to the source you use and read it.  Read the texts, digest, and make a short résumé. If you wih you can include link(s) to the source(s).

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Transcription Factor mapping and prediction

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Modified: 29 March 2020, 7:52 PM   User: Karen Odin  → 

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EMSA (electrophoretic mobility shift assay) or gel shift: technique 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