Purification of Ribosome-Nascent-Chain Complex for Ribosome Profiling and Selective Ribosome Profiling

Hagit Bar-Yosef; Johannes Venezian; Kevin Klann; Ayala Shiber

doi:https://doi.org/10.1007/978-1-0716-2257-5_11

Purification of Ribosome-Nascent-Chain Complex for Ribosome Profiling and Selective Ribosome Profiling

Hagit Bar-Yosef, Johannes Venezian, Kevin Klann, Ayala Shiber

Biology

Technion - Israel Institute of Technology

Research output: Contribution to journal › Article › peer-review

Abstract

Selective Ribosome Profiling (SeRP) is an emerging methodology, developed to capture cotranslational interactions in vivo. To date, SeRP is the only method that can directly capture, in near-codon resolution, ribosomes in action. Thus, SeRP allows us to study the mechanisms of protein synthesis and the network of protein-protein interactions that are formed already during synthesis. Here we report, in detail, the protocol for purification of ribosome- and Nascent-Chain associated factors, followed by isolation of ribosome-protected mRNA footprints, cDNA library generation and subsequent data analysis.

Original language	English
Pages (from-to)	179-193
Number of pages	15
Journal	Methods in molecular biology (Clifton, N.J.)
Volume	2477
DOIs	https://doi.org/10.1007/978-1-0716-2257-5_11
State	Published - 2022

Keywords

Affinity purification
Cotranslational interactions
Nascent-chain
Ribosome
Ribosome occupancy

All Science Journal Classification (ASJC) codes

Molecular Biology
Genetics

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https://doi.org/10.1007/978-1-0716-2257-5_11

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title = "Purification of Ribosome-Nascent-Chain Complex for Ribosome Profiling and Selective Ribosome Profiling",

abstract = "Selective Ribosome Profiling (SeRP) is an emerging methodology, developed to capture cotranslational interactions in vivo. To date, SeRP is the only method that can directly capture, in near-codon resolution, ribosomes in action. Thus, SeRP allows us to study the mechanisms of protein synthesis and the network of protein-protein interactions that are formed already during synthesis. Here we report, in detail, the protocol for purification of ribosome- and Nascent-Chain associated factors, followed by isolation of ribosome-protected mRNA footprints, cDNA library generation and subsequent data analysis.",

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T1 - Purification of Ribosome-Nascent-Chain Complex for Ribosome Profiling and Selective Ribosome Profiling

AU - Bar-Yosef, Hagit

AU - Venezian, Johannes

AU - Klann, Kevin

AU - Shiber, Ayala

PY - 2022

Y1 - 2022

N2 - Selective Ribosome Profiling (SeRP) is an emerging methodology, developed to capture cotranslational interactions in vivo. To date, SeRP is the only method that can directly capture, in near-codon resolution, ribosomes in action. Thus, SeRP allows us to study the mechanisms of protein synthesis and the network of protein-protein interactions that are formed already during synthesis. Here we report, in detail, the protocol for purification of ribosome- and Nascent-Chain associated factors, followed by isolation of ribosome-protected mRNA footprints, cDNA library generation and subsequent data analysis.

AB - Selective Ribosome Profiling (SeRP) is an emerging methodology, developed to capture cotranslational interactions in vivo. To date, SeRP is the only method that can directly capture, in near-codon resolution, ribosomes in action. Thus, SeRP allows us to study the mechanisms of protein synthesis and the network of protein-protein interactions that are formed already during synthesis. Here we report, in detail, the protocol for purification of ribosome- and Nascent-Chain associated factors, followed by isolation of ribosome-protected mRNA footprints, cDNA library generation and subsequent data analysis.

KW - Affinity purification

KW - Cotranslational interactions

KW - Nascent-chain

KW - Ribosome

KW - Ribosome occupancy

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DO - https://doi.org/10.1007/978-1-0716-2257-5_11

M3 - مقالة

C2 - 35524118

SN - 1064-3745

VL - 2477

SP - 179

EP - 193

JO - Methods in molecular biology (Clifton, N.J.)

JF - Methods in molecular biology (Clifton, N.J.)

ER -

Purification of Ribosome-Nascent-Chain Complex for Ribosome Profiling and Selective Ribosome Profiling

Abstract

Keywords

All Science Journal Classification (ASJC) codes

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