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Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells
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Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Article DOI: 10.3791/62639-v • 14:29 min • December 25th, 2021
December 25th, 2021 •
*1, *1, 2, 2, 1,3, 1
1Division of Genome Sciences and Cancer, The John Curtin School of Medical Research, The Australian National University, 2Centre for Advanced Microscopy, The Australian National University, 3Victor Chang Cardiac Research Institute
* These authors contributed equally

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Summary
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We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

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Rapid In Vivo Fixation Isolation Of Translational Complexes Eukaryotic Cells Environmental Conditions Chilling Formaldehyde Fixation Quenching Reaction Primary Amino Group-containing Compounds Glycine Tryps Frozen Storage Mechanical Lysis Chemical Lysis Ribosomal Fractions Molecular Size Sedimentation Properties Affinity Markers Immuno Assisted Purification Detection Electron Microscopy Crosslinked Deep Blocking RNA Amplification Hybridization-based Analyses RNA Sequencing Mass Spectrometry Yeast Culture Orbital Shaker Optical Density Peptone Media Dextrose Media Preparative Centrifuge
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