PMID- 34458400
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20220806
IS  - 2331-8325 (Electronic)
IS  - 2331-8325 (Linking)
VI  - 11
IP  - 15
DP  - 2021 Aug 5
TI  - Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and 
      Tissues Using Ribo Mega-SEC.
PG  - e4106
LID - 10.21769/BioProtoc.4106 [doi]
LID - e4106
AB  - Polysome profile analysis is a popular method for separating polysomes and 
      ribosomal subunits and is typically achieved using a sucrose density gradient 
      (SDG). This has remained the gold standard method since ribosomes were first 
      discovered; however, this method is time-consuming and requires multiple steps 
      from making the gradient and long ultracentrifugation to collecting and analyzing 
      the fractions. Each of these steps in the SDG workflow can introduce potential 
      technical variation that affects the reproducibility of gradient profiles between 
      samples. To address these limitations, we have developed a flexible, alternative 
      approach for analyzing polysomes and ribosomal subunits based on size-exclusion 
      chromatography (SEC), termed 'Ribo Mega-SEC.' In comparison with the SDG method, 
      Ribo Mega-SEC involves a single step using ultra-high-performance liquid 
      chromatography (uHPLC). The entire workflow, from injecting the lysate to 
      collecting the fractions, can be performed in as little as 15 min, with high 
      reproducibility. By varying the pore size of the SEC column, polysomes and 
      ribosomal subunits can be separated using extracts from either human or mouse 
      cultured cell lines or from tissue samples, Drosophila embryos, or budding yeast. 
      The resulting separated fractions are suitable for analysis using a wide range of 
      subsequent analytical techniques including mass spectrometry (MS)-based 
      proteomics, RNA-Seq, electron microscopy (EM), and multiple biochemical assays.
CI  - Copyright (c) The Authors; exclusive licensee Bio-protocol LLC.
FAU - Yoshikawa, Harunori
AU  - Yoshikawa H
AD  - Centre for Gene Regulation and Expression, School of Life Sciences, University of 
      Dundee, Dundee, United Kingdom.
AD  - Division of Cell Signalling, Fujii Memorial Institute of Medical Sciences, 
      Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan.
FAU - Sundaramoorthy, Ramasubramanian
AU  - Sundaramoorthy R
AD  - Centre for Gene Regulation and Expression, School of Life Sciences, University of 
      Dundee, Dundee, United Kingdom.
FAU - Mariyappa, Daniel
AU  - Mariyappa D
AD  - Centre for Gene Regulation and Expression, School of Life Sciences, University of 
      Dundee, Dundee, United Kingdom.
FAU - Jiang, Hao
AU  - Jiang H
AD  - Centre for Gene Regulation and Expression, School of Life Sciences, University of 
      Dundee, Dundee, United Kingdom.
FAU - Lamond, Angus I
AU  - Lamond AI
AD  - Centre for Gene Regulation and Expression, School of Life Sciences, University of 
      Dundee, Dundee, United Kingdom.
LA  - eng
PT  - Journal Article
DEP - 20210805
PL  - United States
TA  - Bio Protoc
JT  - Bio-protocol
JID - 101635102
PMC - PMC8376616
OTO - NOTNLM
OT  - Polysome
OT  - Polysome profile
OT  - Protein synthesis
OT  - Ribosome
OT  - Size-exclusion chromatography
OT  - Sucrose density gradient
OT  - mRNA translation
COIS- Competing interestsThe authors declare no competing interests.
EDAT- 2021/08/31 06:00
MHDA- 2021/08/31 06:01
PMCR- 2022/08/05
CRDT- 2021/08/30 06:02
PHST- 2021/02/23 00:00 [received]
PHST- 2021/04/19 00:00 [revised]
PHST- 2021/04/19 00:00 [accepted]
PHST- 2021/08/30 06:02 [entrez]
PHST- 2021/08/31 06:00 [pubmed]
PHST- 2021/08/31 06:01 [medline]
PHST- 2022/08/05 00:00 [pmc-release]
AID - e4106 [pii]
AID - 4106 [pii]
AID - 10.21769/BioProtoc.4106 [doi]
PST - epublish
SO  - Bio Protoc. 2021 Aug 5;11(15):e4106. doi: 10.21769/BioProtoc.4106. eCollection 
      2021 Aug 5.