Polysome profiling assays were implemented as previously described with some modifications [38]. Ribosome was extracted from the leaves by grounding in liquid nitrogen. The pulverized tissue were homogenized in pre-chilled ribosome extraction buffer (0.2 M Tris-HCl pH 9.0, 0.2 M KCl, 0.035 M MgCl2, 0.025M EGTA, 50 μg/mL cycloheximide, 50 μg/mL chloramphenicol, 1% Triton X-100, 5 mM DTT, and Protease Inhibitor Cocktail) for 30 min on ice with gentle mixing to ensure thoroughly lysis. The mixture subsequently filtered through two layers of sterile microcloth into an RNA-free tube. The extracts were loaded gently and slowly on top of the sucrose cushion (0.4 M Tris pH 9.0, 0.2 M KCl, 0.005 M EGTA, 0.035 M MgCl2, 1.75 M Sucrose) to avoid mixing the sample with sucrose cushion and centrifuged at 4 °C, 170,000× g (Beckman Ti70 rotor) for 3 h to get the ribosome pellets. The resulting pellet were resuspended in 700 μL of resuspension buffer (0.2 M Tris–HCl pH 9.0, 0.2 mM KCl, 35 mM MgCl2, 25 mM EGTA, 100 μg/mL chloramphenicol, and 50 μg/mL cycloheximide) and incubated on ice for 30 min. The ribosome-containing solutions were loaded onto an 11-mL 20–60% continuous sucrose density gradient and centrifuged at 237,000× g for 4 h at 4 °C (Beckman SW 40 Ti rotor). After ultracentrifugation, the absorbance of 254 nm was continuously monitored to detect the position of the different compositions using a Teledyne Isco Density Gradient Fractionation System (Teledyne Isco, Lincoln, NE, USA) with spectrophotometric detection device.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.