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Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides
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JoVE Journal Biologie
Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

DOI:
10.3791/4027-v

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09:42 min

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June 19, 2012

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,
1Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences,Cleveland State University

Kapitel

  • 00:05Titel
  • 03:31DNA Template Preparation, In vitro Transcription and Translation
  • 05:34Isolation of Nascent Polypeptides and Analysis by Tris-tricine SDS PAGE
  • 07:17Representative RNC Results
  • 07:57Conclusion

Summary

Automatische Übersetzung

Automatische Übersetzung

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Tags

SecM Arrest SequenceRibosome Bound PolypeptidesCo-translational FoldingIsolation TechniqueRNCs (ribosome-nascent Chain Complexes)Protein Folding PathwayConformational AnalysisSecM ProteinSecretion MonitorSecA ATPaseStalling SequencePeptidyl-glycyl-tRNAC-terminal RegionPolypeptide Chain ElongationIn Vivo Studies

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