An easy method for plant polysome profiling - Archive ouverte HAL Access content directly
Journal Articles Journal of visualized experiments : JoVE Year : 2016

An easy method for plant polysome profiling

(1, 2) , (2) , (3) , (2) , (2)
1
2
3

Abstract

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.
Fichier principal
Vignette du fichier
jove-114-54231.pdf (488.98 Ko) Télécharger le fichier
Origin : Publication funded by an institution

Dates and versions

cea-02370683 , version 1 (06-01-2023)

Identifiers

Cite

Cécile Lecampion, Maïna Floris, Jean-Raphael Fantino, Christophe Robaglia, Christophe Laloi. An easy method for plant polysome profiling. Journal of visualized experiments : JoVE, 2016, 114, pp.e54231. ⟨10.3791/54231⟩. ⟨cea-02370683⟩
0 View
0 Download

Altmetric

Share

Gmail Facebook Twitter LinkedIn More