Abstract
The rabbit reticulocyte lysate is a highly efficient system for protein synthesis in vitro, and therefore has been used frequently in studies of translational control. However, when the lysate is fractionated into ribosomes and soluble proteins by centrifugation, there is a severe loss of activity, thereby making the system less suitable for identifying components involved in translational control. We have devised a new method of cell fractionation which employs rapid (20-min) centrifugation to pellet ribosomes. The post-ribosomal supernatant (S-100), high salt-washed ribosomes, and the high salt wash are all required in the reconstituted protein synthesis assay, and greater than 70% of the activity of the unfractionated lysate is attained. Proteins in the high salt wash have been further fractionated by ammonium sulfate precipitation, and the assay system has been used to measure the activities of eIF-4B and eIF-4F. This highly active fractionated lysate system should be useful in measuring the specific activities of translational components and may be appropriate for detecting changes due to covalent modifications such as phosphorylation.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
