Abstract
The Ribo-ELISA was originally developed to elucidate the basis for the ribopuromycylation method (RPM)-based detection of ribosome bound nascent chains. The Ribo-ELISA enables characterization of the translational status of ribosomes, and can be applied to the discovery of super-ribosomal complexes with novel ribosome associated macromolecules that are isolated by physical fractionation in sucrose gradients or other methods.
Keywords: Ribosome, ELISA, Ribopuromycylation
Background
Ribosomes are heterogeneous structures consisting of 40S and 60S subunits that are present in cells as monosomes and polysomes, when multiple ribosomes are bound to a single mRNA. Additionally, translating ribosomes can be associated with multiple molecular complexes that modulate translation. The ribosome ELISA (Enzyme-Linked ImmunoSorbent Assay) enables detection of translating ribosomes by in vitro puromycylation of ribosome associated nascent chains (David et al., 2012). This chemical reaction proceeds spontaneously upon adding puromycin to ribosomes with bound nascent chains. This method quantitates the number of nascent chains present per ribosome and can be used to determine the translational status of monosomes relative to polysomes, and identify ribosomes bound to other macromolecules that alter its sedimentation rate or migration in sizing columns.
Materials and Reagents
Equipment
Software
Procedure
The following procedure describes the first application of Ribo-ELISA (David et al., 2012) which was used to detect puromycin association with ribosomes: at that time it was not clear whether puromycin binding was limited to translating ribosomes. Either emetine or CHX can be used as elongation inhibitors to freeze polysome. Unlike CHX, emetine irreversibly inhibits translation, and need not be maintained throughout the procedure. Day 1:
Day 2:
Data analysis
For data analysis, we previously used GraphPad Prism software. An example is presented in the following paper: David et al., 2012.
Notes
Polysome profile can vary dramatically depending on the cell line and translation rate.
Recipes
Acknowledgments
JWY is supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases. AD benefits from generous funding from Fondation pour la Recherche Médicale, Ligue contre le Cancer and Cancéropôle GSO. The authors declare no conflicts of interest or competing interests.
References
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