Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

An efficient mRNA-dependent translation system from reticulocyte lysates

H R Pelham, R J Jackson

    European Journal of Biochemistry
    |August 1, 1976
    PubMed
    Summary

    Researchers developed a method to create an mRNA-dependent protein synthesis system from rabbit reticulocyte cell-free extract. This system efficiently translates various mRNAs into full-sized proteins, proving highly sensitive and reliable for molecular biology research.

    Related Concept Videos

    You might also read

    Related Articles

    Articles linked to this work by shared authors, journal, and citation graph.

    Sort by
    Same author

    Correction to: short and long-term acceptability and efficacy of extended-release cornstarch in the hepatic glycogen storage diseases: results from the Glyde study.

    Orphanet journal of rare diseases·2024
    Same author

    Short and long-term acceptability and efficacy of extended-release cornstarch in the hepatic glycogen storage diseases: results from the Glyde study.

    Orphanet journal of rare diseases·2024
    Same author

    A hierarchical role of IL-25 in ILC development and function at the lung mucosae following viral-vector vaccination.

    Vaccine: X·2019
    Same author

    Viral vector and route of administration determine the ILC and DC profiles responsible for downstream vaccine-specific immune outcomes.

    Vaccine·2019
    Same author

    Extensive Fetal Congenital Subcutaneous Mixed Venous Lymphatic Lesion: Prenatal Diagnosis and Postnatal Management.

    AJP reports·2015
    Same author

    Unique IL-13Rα2-based HIV-1 vaccine strategy to enhance mucosal immunity, CD8(+) T-cell avidity and protective immunity.

    Mucosal immunology·2013

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Cell-Free Protein Synthesis

    Background:

    • Rabbit reticulocyte lysate is a common cell-free system for protein synthesis.
    • Endogenous mRNA in the lysate can interfere with studies of specific mRNA translation.
    • A need exists for a depleted lysate system that is mRNA-dependent and highly efficient.

    Purpose of the Study:

    • To develop a simple method for converting standard rabbit reticulocyte lysate into an mRNA-dependent protein synthesis system.
    • To characterize the efficiency and sensitivity of the modified system.
    • To assess the system's ability to translate various mRNA species, including viral RNA.

    Main Methods:

    • Preincubation of lysate with CaCl2 and micrococcal nuclease to degrade endogenous mRNA.

    Related Experiment Videos

  • Addition of excess ethyleneglycol-bis(2-aminoethylether)-N,N'-tetraacetic acid (EGTA) to chelate Ca2+ and inactivate nuclease.
  • Assay of protein synthesis activity upon addition of exogenous mRNA (e.g., globin mRNA).
  • Analysis of translation products for size and integrity.

    • Main Results:

    • The modified lysate exhibited negligible endogenous amino acid incorporation.
    • Addition of globin mRNA restored 75% of the original lysate's protein synthesis activity.
    • The system demonstrated high efficiency in mRNA utilization and sensitivity.
    • No residual nuclease activity was detected, and tRNA remained functionally unimpaired.
    • Efficient translation of various mRNAs (up to 200,000 MW) and plant viral RNAs was achieved.
    • No accumulation of incomplete protein products was observed.

    Conclusions:

    • A simple and effective method was established to create an mRNA-dependent protein synthesis system from rabbit reticulocyte lysate.
    • The developed system is highly efficient, sensitive, and suitable for translating diverse mRNA species, including viral RNA.
    • This system provides a valuable tool for studying mRNA translation and protein synthesis in molecular biology research.