Fluorescein-Based Electrophoretic Mobility Shift Assay.

Electrophoretic Mobility Shift Assay Fluorescein Protein Binding RNA / metabolism RNA-Binding Proteins
EMSA Fluorescein Viroid

Journal

Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969

Informations de publication

Date de publication:
2022
Historique:
entrez: 30 11 2021
pubmed: 1 12 2021
medline: 19 1 2022
Statut: ppublish

Résumé

RNA-protein complexes are functionally important in biology. Electrophoretic mobility shift assays (EMSA) have been widely used to study the molecular basis of protein-RNA interactions. Previous methods for EMSA mostly relied on radioactive RNA substrates, raising health and environmental concerns. Here, we describe a method based on fluorescein-labeled RNA for EMSA. In addition, we simplified the protocol to efficiently purify RNA-binding proteins from bacterial expression systems.

Identifiants

DOI: 10.1007/978-1-0716-1464-8_12 PMID: 34845691
pubmed: 34845691
doi: 10.1007/978-1-0716-1464-8_12
doi:

Substances chimiques

RNA-Binding Proteins 0
RNA 63231-63-0
Fluorescein TPY09G7XIR

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

133-140

Informations de copyright

© 2022. Springer Science+Business Media, LLC, part of Springer Nature.

Références

Baltz AG, Munschauer M, Schwanhausser B et al (2012) The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol Cell 46:674–690
doi: 10.1016/j.molcel.2012.05.021
Castello A, Fischer B, Eichelbaum K et al (2012) Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149:1393–1406
doi: 10.1016/j.cell.2012.04.031
Hentze MW, Castello A, Schwarzl T et al (2018) A brave new world of RNA-binding proteins. Nat Rev Mol Cell Biol 19:327–341
doi: 10.1038/nrm.2017.130
Hellman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc 2:1849–1861
doi: 10.1038/nprot.2007.249
Ryder SP, Recht MI, Williamson JR (2008) Quantitative analysis of protein-RNA interactions by gel mobility shift. Methods Mol Biol 488:99–115
doi: 10.1007/978-1-60327-475-3_7
Ding B (2009) The biology of viroid-host interactions. Annu Rev Phytopathol 47:105–131
doi: 10.1146/annurev-phyto-080508-081927
Flores R, Gago-Zachert S, Serra P et al (2014) Viroids: survivors from the RNA world? Annu Rev Microbiol 68:395–414
doi: 10.1146/annurev-micro-091313-103416
Flores R, Minoia S, Carbonell A et al (2015) Viroids, the simplest RNA replicons: how they manipulate their hosts for being propagated and how their hosts react for containing the infection. Virus Res 209:136–145
doi: 10.1016/j.virusres.2015.02.027
Layat E, Probst AV, Tourmente S (2013) Structure, function and regulation of transcription factor IIIA: from Xenopus to Arabidopsis. Biochim Biophys Acta 1829:274–282
doi: 10.1016/j.bbagrm.2012.10.013
Fu Y, Bannach O, Chen H et al (2009) Alternative splicing of anciently exonized 5S rRNA regulates plant transcription factor TFIIIA. Genome Res 19:913–921
doi: 10.1101/gr.086876.108
Hammond MC, Wachter A, Breaker RR (2009) A plant 5S ribosomal RNA mimic regulates alternative splicing of transcription factor IIIA pre-mRNAs. Nat Struct Mol Biol 16:541–549
doi: 10.1038/nsmb.1588
Layat E, Cotterell S, Vaillant I et al (2012) Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development. Plant J 71:35–44
doi: 10.1111/j.1365-313X.2012.04948.x
Eiras M, Nohales MA, Kitajima EW et al (2011) Ribosomal protein L5 and transcription factor IIIA from Arabidopsis thaliana bind in vitro specifically Potato spindle tuber viroid RNA. Arch Virol 156:529–533
doi: 10.1007/s00705-010-0867-x
Wang Y, Qu J, Ji S et al (2016) A land plant-specific transcription factor directly enhances transcription of a pathogenic noncoding RNA template by DNA-dependent RNA polymerase II. Plant Cell 28:1094–1107
doi: 10.1105/tpc.16.00100
Ottonello S, Ballabeni A, Soncini C et al (1994) High level expression in E. coli and purification of yeast transcription factor IIIA. Biochem Biophys Res Commun 203:1217–1223
doi: 10.1006/bbrc.1994.2312
Clarke S (1981) Direct renaturation of the dodecyl sulfate complexes of proteins with Triton X-100. Biochim Biophys Acta 670:195–202
doi: 10.1016/0005-2795(81)90009-X
Tao H, Liu W, Simmons BN et al (2010) Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS. BioTechniques 48:61–64
doi: 10.2144/000113304

Auteurs

Shachinthaka D Dissanayaka Mudiyanselage (SDD)

Department of Biological Sciences, Mississippi State University, Mississippi State, MS, USA.

Ying Wang (Y)

Department of Biological Sciences, Mississippi State University, Mississippi State, MS, USA. wang@biology.msstate.edu.

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Classifications MeSH

questionsmedicales.fr › Techniques d'investigation › Techniques de chimie analytique › Test de retard de migration électrophorétique › Fluorescein-Based Electrophoretic Mobility Shift Assay.
questionsmedicales.fr › Composés polycycliques › Spiranes › Fluorescéines › Fluorescéine › Fluorescein-Based Electrophoretic Mobility Shift Assay.
questionsmedicales.fr › Métabolisme › Liaison aux protéines › Fluorescein-Based Electrophoretic Mobility Shift Assay.
questionsmedicales.fr › Acides nucléiques, nucléotides et nucléosides › Acides nucléiques › ARN › Fluorescein-Based Electrophoretic Mobility Shift Assay.
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Nucléoprotéines › Protéines de liaison à l'ARN › Fluorescein-Based Electrophoretic Mobility Shift Assay.