Journal
/
Biology
/
Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides
JoVE Journal
Biology
A subscription to JoVE is required to view this content.  Sign in or start your free trial.
JoVE Journal Biology
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

•

09:42 min

•

June 19, 2012

•

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

Chapters

  • 00:05Title
  • 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

Automatic Translation

Automatic Translation

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

Article

Embed

ADD TO PLAYLIST

Usage Statistics

Related Videos

check_url/4027?article_type=v

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces
check_url/4027?article_type=v

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets
check_url/4027?article_type=v

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles
check_url/4027?article_type=v

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
check_url/4027?article_type=v

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism
check_url/4027?article_type=v

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique
check_url/4027?article_type=v

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types
check_url/4027?article_type=v

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model
check_url/4027?article_type=v

A High Output Method to Isolate Cerebral Pericytes from Mouse
check_url/4027?article_type=v

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

Read Article