Analysis of plant ribosomes with asymmetric flow field-flow fractionation Field-Flow Fractionation

Leena Pitkänen, Päivi Tuomainen, Katri Eskelin*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

6 Citations (Scopus)

Abstract

Ribosome profiling is a technique used to separate ribosomal subunits, 80S ribosomes (monosomes), and polyribosomes (polysomes) from other RNA-protein complexes. It is traditionally performed in sucrose gradients. In this study, we used asymmetric flow field-flow fractionation (AsFlFFF) to characterize ribosome profiles of Nicotiana benthamiana plants. With the optimized running conditions, we were able to separate free molecules from ribosomal subunits and intact ribosomes. We used various chemical and enzymatic treatments to validate the positions of subunits, monosomes, and polysomes in the AsFlFFF fractograms. We also characterized the protein and RNA content of AsFlFFF fractions by gel electrophoresis and western blotting. The reverse transcription polymerase chain reaction (RT-PCR) analysis showed that ribosomes remained bound to messenger RNAs (mRNAs) during the analysis. Therefore, we conclude that AsFlFFF can be used for ribosome profiling to study the mRNAs that are being translated. It can also be used to study the protein composition of ribosomes that are active in translation at that particular moment.

Original languageEnglish
Pages (from-to)1629-1637
Number of pages9
JournalANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume406
Issue number6
DOIs
Publication statusPublished - 1 Jan 2014
MoE publication typeA1 Journal article-refereed

Keywords

  • Asymmetric flow field-flow fractionation
  • Plants
  • Polysomes
  • Ribosomal subunits
  • Ribosomes

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