Electrophoretic Mobility Shift Assay of
CTD Code
Kinase
Phosphorylation
RNA polymerase II
Transcription
mRNA
Journal
Bio-protocol
ISSN: 2331-8325
Titre abrégé: Bio Protoc
Pays: United States
ID NLM: 101635102
Informations de publication
Date de publication:
20 Jun 2020
20 Jun 2020
Historique:
received:
30
12
2019
revised:
29
04
2020
accepted:
29
04
2020
entrez:
4
3
2021
pubmed:
5
3
2021
medline:
5
3
2021
Statut:
epublish
Résumé
Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques exist to identify and quantify the degree of CTD phosphorylation, including phosphorylation-specific antibodies and mass spectrometry. Electrophoretic mobility shift assays (EMSAs) have been utilized since the discovery of CTD phosphorylation and continue to represent a simple, direct, and widely applicable approach for qualitatively monitoring CTD phosphorylation. We present a standardized method for EMSA analysis of recombinant GST-CTD substrates phosphorylated by a variety of CTD kinases. Strategies to analyze samples under both denatured/reduced and semi-native conditions are provided. This method represents a simple, direct, and reproducible means to monitor CTD phosphorylation in recombinant substrates utilizing equipment common to molecular biology labs and readily applicable to downstream analyses including immunoblotting and mass spectrometry.
Identifiants
pubmed: 33659319
doi: 10.21769/BioProtoc.3648
pii: e3648
pmc: PMC7842589
doi:
Types de publication
Journal Article
Langues
eng
Pagination
e3648Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM104896
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM125882
Pays : United States
Informations de copyright
Copyright © The Authors; exclusive licensee Bio-protocol LLC.
Déclaration de conflit d'intérêts
Competing interestsThe authors declare no competing financial interests.
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