Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)
نویسندگان
چکیده
منابع مشابه
Electrophoretic Mobility Shift Assay (EMSA).
Transcriptional regulation of gene expression is controlled through the binding of sequence-specific DNA-binding proteins (transcription factors) to the regulatory regions of genes. The exact gene expression program of a cell is determined by the spectrum of transcription factors present with the nucleus of a cell. The presence of these factors is dependent upon the cell type being examined and...
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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 ...
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Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages o...
متن کاملDesign of a fluorescent electrophoretic mobility shift assay improved for the quantitative and multiple analysis of protein-DNA complexes.
We describe a protocol for the fluorescent electrophoretic mobility shift assay improved for the quantitative analysis of protein-DNA complexes. Fluorescent-labeled oligonucleotide probes incubated with nuclear proteins were followed by electrophoresis. The signals for protein-DNA complexes were measured and normalized with fluorescent-labeled marker using fragment analysis software. This assay...
متن کاملElectrophoretic mobility shift scanning using an automated infrared DNA sequencer.
Electrophoretic mobility shift assay (EMSA) is widely used in the study of sequence-specific DNA-binding proteins, including transcription factors and mismatch binding proteins. We have established a non-radioisotope-based protocol for EMSA that features an automated DNA sequencer with an infrared fluorescent dye (IRDye) detection unit. Our modification of the elec- trophoresis unit, which incl...
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ژورنال
عنوان ژورنال: Journal of Visualized Experiments
سال: 2016
ISSN: 1940-087X
DOI: 10.3791/54093