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EMSA Electrophoretic Mobility Shift Assay

This Electrophoretic Mobility Shift Assay site is your portal for all things related to the EMSA technique and its analysis. We provide you with all the EMSA protocols, transcriptional factor databases, and DNA RNA binding protein bioinformatic, and mobility shift products you will need for your research endeavours.

 

You will also find the latest published papers emerging from this exciting field, and the latest news on DNA and RNA binding proteins.

Electrophoretic Mobility Shift Assay Articles

EMSA The best background to learn about the electrophoretic mobility shift assay in our encyclopedia.

DNA EMSA

DNA EMSA

EMSA Abbreviation Meaning.

Related EMSA Methods

DNA Footprinting

Related Mobility Shift Topics

DNA Binding Proteins

RNA Binding Proteins

Transcription Factors

EMSA Troubleshooting

EMSA Troubleshooting

EMSA Forum Topics

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EMSA Research Publications

IDENTIFICATION OF KSHV LANA REGIONS IMPORTANT FOR EPISOME SEGREGATION, REPLIC...

IDENTIFICATION OF KSHV LANA REGIONS IMPORTANT FOR EPISOME SEGREGATION, REPLICATION AND PERSISTENCE.

J Virol. 2013 Sep 4;

Authors: De León Vázquez E, Carey VJ, Kaye KM

Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) is an 1162 amino acid protein that mediates maintenance of episomal viral genomes in latently infected cells. The two central components of episome persistence are DNA replication with each cell division, and segregation of DNA to progeny nuclei. LANA self-associates to bind KSHV terminal repeat (TR) DNA and mediate its replication. LANA also simultaneously binds to TR DNA and mitotic chromosomes to mediate segregation of episomes to daughter nuclei. N-terminal LANA binds histones H2A/H2B to attach to mitotic chromosomes while C-terminal LANA binds TR DNA and also associates with chromosomes. Both N- and C-terminal LANA are essential for episome persistence. We recently showed that deletion of all internal LANA sequence results in highly deficient episome maintenance. Here, we assess independent internal LANA regions for effects on episome persistence. We generated a panel of LANA mutants that included deletions in the large internal repeat region and in the unique internal sequence. All mutants contained the essential N- and C-terminal regions, and as expected, all maintained the ability to associate with mitotic chromosomes in a wild-type fashion and bind TR DNA as assessed by EMSA. Deletion of the internal regions did not reduce LANA half-life. Notably, deletions within either the repeat elements or unique sequence resulted in deficiencies in DNA replication. However, only the unique internal sequence exerted effects on LANA's ability to retain GFP expression from TR containing episomes deficient for DNA replication, consistent with a role in episome segregation; this region did not independently associate with mitotic chromosomes. All mutants were deficient for episome persistence, and the deficiencies ranged from minor to severe. Those mutants deficient for DNA replication and containing deletions within the unique internal sequence had the most severe deficits. These data suggest that internal LANA regions exert critical roles in LANA mediated DNA replication, segregation, and episome persistence, likely by acting through interactions with key host cell factors.

PMID: 24006437 [PubMed - as supplied by publisher]


Recruitment of Specificity Protein 1 by CpG hypomethylation upregulates Na+-K...

Recruitment of Specificity Protein 1 by CpG hypomethylation upregulates Na+-K+-2Cl- cotransporter 1 in hypertensive rats.

J Hypertens. 2013 Jul;31(7):1406-13

Authors: Cho HM, Lee HA, Kim HY, Lee DY, Kim IK

Abstract
OBJECTIVES: Promoter hypomethylation leads to upregulation of Na-K-2Cl cotransporter 1 (NKCC1) in the spontaneously hypertensive rat (SHR). We hypothesized that recruitment of Specificity Protein 1 (Sp1) by CpG hypomethylation would result in upregulation of Na-K-2Cl cotransporter 1 in hypertensive rats.
METHODS: Sham-operated Wistar-Kyoto (WKY) rats (sham) and angiotensin II (Ang II)-infused WKY rats, as well as SHRs, were used in this study. We performed real-time PCR and western blot for determination of the expression levels of Nkcc1 mRNA and protein, respectively, and bisulphite sequencing for determination of the methylation status of the proximal promoter; an assay kit was used for assessment of the activity of DNA methyltransferase (DNMT), and the electrophoretic mobility shift assay (EMSA) was used for assessment of binding of Sp1 to cis-element, and promoter function was assessed using the luciferase assay.
RESULTS: Both Ang II-infused WKY rats and SHRs showed higher expression of Nkcc1 mRNA and protein and less DNA methylation, compared with sham. CpG methylation at Sp1 response elements interfered with binding of Sp1, resulting in disabled promoter activity. Both types of hypertensive rats showed hypomethylation of CpG dinucleotides in Sp1 response elements in accordance with the decrease of DNMT activity. DNMT3b and MeCP2 were highly recruited to the more methylated promoter of normotensive rats, whereas the CXXC finger protein 1 (Cfp1), Sp1 and RNA polymerase II were highly recruited to the less methylated promoter of hypertensive rats.
CONCLUSION: Our results indicate that recruitment of Sp1 by CpG hypomethylation leads to upregulation of Na-K-2Cl cotransporter 1 in hypertensive rats.

PMID: 24006039 [PubMed - in process]


Recognition of intermolecular G-quadruplexes by full length nucleophosmin. Ef... Related Articles

Recognition of intermolecular G-quadruplexes by full length nucleophosmin. Effect of a leukaemia-associated mutation.

FEBS Lett. 2013 Jul 11;587(14):2254-9

Authors: Bañuelos S, Lectez B, Taneva SG, Ormaza G, Alonso-Mariño M, Calle X, Urbaneja MA

Abstract
Nucleophosmin (NPM) is a nucleolar protein involved in ribosome biogenesis. NPM1 gene is frequently mutated in acute myeloid leukaemia (AML), correlating with aberrant cytoplasmic localization of the protein. NPM attachment to the nucleolus in physiological conditions probably depends on binding to nucleic acids, and this recognition could be altered in AML. NPM associates to guanine-rich DNA sequences, able to fold as "G-quadruplexes". We have analyzed the interaction of pentameric, full length NPM with G-rich oligonucleotides, finding that the protein binds preferentially high-order G-quadruplexes. AML-associated mutation significantly hampers DNA binding, pointing to a possible mechanism contributing to pathological mislocalization of NPM.

PMID: 23742937 [PubMed - indexed for MEDLINE]


 

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