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Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells
JoVE 杂志
生物学
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JoVE 杂志 生物学
Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

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

DOI:
10.3791/62639-v

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14:29 min

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December 25, 2021

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, , , , ,
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

Chapters

  • 00:10Introduction
  • 01:06Yeast Cell Culture and FIxation
  • 03:58Yeast Cell Disruption and Cytosol Collection
  • 05:45Mammalian Cell Culture and Fixation
  • 09:40Mammalian Cell Disruption and Cytosol Collection
  • 11:02Results
  • 13:41Conclusion

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.

Tags

Keywords: In Vivo FixationTranslational ComplexesEukaryotic CellsRapid ChillingFormaldehyde FixationGlycine QuenchingCell LysisRibosomal FractionsAffinity PurificationRNA AnalysisMass SpectrometryYeast CultureExponential Growth PhaseFormaldehyde ConcentrationFixation TimeCell PelletingGlycine Buffer

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