Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

Hellman, Lance M.; Fried, Mike

doi:10.1038/nprot.2007.249

Cited by 915 publications

(690 citation statements)

References 114 publications

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“…Conditions for EMSA have been previously described. 64 α 32 P-UTP-labeled p53 1-251 IRES (25 fmol) was incubated with 300 and 600 ng of His-SMAR1 protein at 30°C for 30 min. Loading dye was added and the protein-nucleotidyl complex was resolved on a 4% (60 : 1) polyacrylamide gel at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Reversible induction of translational isoforms of p53 in glucose deprivation

et al. 2015

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Tumor suppressor protein p53 is a master transcription regulator, indispensable for controlling several cellular pathways. Earlier work in our laboratory led to the identification of dual internal ribosome entry site (IRES) structure of p53 mRNA that regulates translation of full-length p53 and Δ40p53. IRES-mediated translation of both isoforms is enhanced under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum stress, oncogene-induced senescence and cancer. In this study, we addressed nutrient-mediated translational regulation of p53 mRNA using glucose depletion. In cell lines, this nutrient-depletion stress relatively induced p53 IRES activities from bicistronic reporter constructs with concomitant increase in levels of p53 isoforms. Surprisingly, we found scaffold/matrix attachment region-binding protein 1 (SMAR1), a predominantly nuclear protein is abundant in the cytoplasm under glucose deprivation. Importantly under these conditions polypyrimidine-tractbinding protein, an established p53 ITAF did not show nuclear-cytoplasmic relocalization highlighting the novelty of SMAR1-mediated control in stress. In vivo studies in mice revealed starvation-induced increase in SMAR1, p53 and Δ40p53 levels that was reversible on dietary replenishment. SMAR1 associated with p53 IRES sequences ex vivo, with an increase in interaction on glucose starvation. RNAi-mediated-transient SMAR1 knockdown decreased p53 IRES activities in normal conditions and under glucose deprivation, this being reflected in changes in mRNAs in the p53 and Δ40p53 target genes involved in cell-cycle arrest, metabolism and apoptosis such as p21, TIGAR and Bax. This study provides a new physiological insight into the regulation of this critical tumor suppressor in nutrient starvation, also suggesting important functions of the p53 isoforms in these conditions as evident from the downstream transcriptional target activation. Cell Death and Differentiation (2015) 22, 1203-1218; doi:10.1038/cdd.2014.220; published online 27 February 2015 p53 is a master transcription factor and tumor suppressor. Apart from post-translational modifications of p53 and concomitant protein-protein interactions of p53 with diverse factors, translational control of p53 mRNA also plays an important role under stress conditions. 1 p53 and its N-terminally truncated isoform Δ40p53 (also known as ΔN-p53 or p53/47) are translated by internal ribosome entry site (IRES)-mediated translation initiation from the same mRNA under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum (ER) stress, oncogene-induced senescence and cancer. [2][3][4][5][6][7] Thus, p53 mRNA has a dual IRES structure. 8 For their function, these IRESs rely on IRES trans-acting factors (ITAFs). Polypyrimidine-tract-binding protein (PTB) was shown to have differential affinity for the two IRESs of p53 and regulated p53 IRES functions by translocating from nucleus to cytoplasm on doxorubicin-induced DNA damage. 3 This PTB-med...

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Section: Methodsmentioning

confidence: 99%

Reversible induction of translational isoforms of p53 in glucose deprivation

et al. 2015

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“…Causative inferences can then be made by applying mutagenesis to the candidate regulatory regions. Electrophoretic Mobility Shift Assays (Hellman & Fried, 2007). are also often used to screen nuclear extract or DNA sequences for specific protein-DNA binding activity.…”

Section: Investigating Non-coding Locimentioning

confidence: 99%

Translating Lung Function Genome-Wide Association Study (GWAS) Findings

Kheirallah

Miller

Hall

et al. 2016

Advances in Genetics

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“…An excellent example is the supershift assay in which the identity of a DNA-binding protein is confirmed only when a protein-specific antibody reduces the electrophoretic mobility of a protein-DNA interaction complex. 30 A significant advantage of such a gel shift procedure over other protein-DNA interaction detection methods is its ability to distinguish single from multimeric forms of bound protein and to immediately relate this information to the respective DNA bait. For example, using supershift assays, Tantin and colleagues 31 determined that DNA baits were more likely to induce di-or multimerization of the TF Oct-4 when they contained at least three Oct-4 half sites.…”

Section: Dna-binding Protein Identification Antibodiesmentioning

confidence: 99%

DNA-centered approaches to characterize regulatory protein–DNA interaction complexes

Simicevic

Deplancke

2010

Mol. BioSyst.

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Gene regulation is mediated by site-specific DNA-binding proteins or transcription factors (TFs), which form protein complexes at regulatory loci either to activate or repress the expression of a target gene. The study of the dynamic properties of these regulatory DNA-binding complexes has so far been dominated by protein-centered methodologies, aiming to characterize the DNA-binding behavior of one specific protein at a time. With the emerging evidence for a role of DNA in allosterically influencing DNA-binding protein complex formation, there is renewed interest in DNA-centered approaches to capture protein complexes on defined regulatory loci and to correlate changes in their composition with alterations in target gene expression. In this review, we present the current state-of-the-art in such DNA-centered approaches and evaluate recent technological improvements in the purification as well as in the identification of regulatory DNA-binding protein complexes within or outside their biological context. Finally, we suggest possible areas of improvement and assess the putative impact of DNA-centered methodologies on the gene regulation field for the forthcoming years.

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Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

Cited by 915 publications

References 114 publications

Reversible induction of translational isoforms of p53 in glucose deprivation

Reversible induction of translational isoforms of p53 in glucose deprivation

Translating Lung Function Genome-Wide Association Study (GWAS) Findings

DNA-centered approaches to characterize regulatory protein–DNA interaction complexes

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