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RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse
JoVE Journal
Developmental Biology
This content is Free Access.
JoVE Journal Developmental Biology
RiboTag Immunoprecipitation in the Germ Cells of the Male Mouse
DOI:

10:00 min

March 04, 2020

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Chapters

  • 00:05Introduction
  • 00:56Preparation of Solutions
  • 01:57Preparation of Tissue
  • 02:49Homogenization of Sample
  • 04:03Equilibration of Beads
  • 04:59Preclearing Sample
  • 05:56Incubation of Antibody and Beads
  • 06:29Washing of Beads
  • 07:21RNA Extraction
  • 07:54Results: Negative Controls, Reagents Impact, and a Mutant Model
  • 09:09Conclusion

Summary

Automatic Translation

Here, we describe the immunoprecipitation of ribosomes and associated RNA from select populations of adult male mouse germ cells using the RiboTag system. Strategic breeding and careful immunoprecipitation result in clean, reproducible results that inform on the germ cell translatome and provide insight into the mechanisms of mutant phenotypes.

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