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Verfasst von:Inchaustegui Gil, Diana Patricia [VerfasserIn]   i
 Clayton, Christine [VerfasserIn]   i
Titel:Purification of messenger ribonucleoprotein particles via a tagged nascent polypeptide
Verf.angabe:Diana P. Inchaustegui Gil, Christine Clayton
E-Jahr:2016
Jahr:2016 Jan 25
Umfang:13 S.
Fussnoten:Gesehen am 10.06.2020
Titel Quelle:Enthalten in: PLOS ONE
Ort Quelle:San Francisco, California, US : PLOS, 2006
Jahr Quelle:2016
Band/Heft Quelle:11(2016,1) Artikel-Nummer e0148131, 13 Seiten
ISSN Quelle:1932-6203
Abstract:The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA.
DOI:doi:10.1371/journal.pone.0148131
URL:Bitte beachten Sie: Dies ist ein Bibliographieeintrag. Ein Volltextzugriff für Mitglieder der Universität besteht hier nur, falls für die entsprechende Zeitschrift/den entsprechenden Sammelband ein Abonnement besteht oder es sich um einen OpenAccess-Titel handelt.

Volltext ; Verlag: https://doi.org/10.1371/journal.pone.0148131
 Volltext: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726818/
 DOI: https://doi.org/10.1371/journal.pone.0148131
Datenträger:Online-Ressource
Sprache:eng
K10plus-PPN:1700315307
Verknüpfungen:→ Zeitschrift

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