Abstract
Polysome profiling by sucrose density gradient centrifugation is commonly used to study the overall degree of translation (messenger RNA to protein synthesis). Traditionally, the method begins with synthesis of a 5-10 mL sucrose gradient onto which 0.5-1 mL of cell extract is layered and centrifuged at high speed for 3-4 h in a floor-model ultracentrifuge. After centrifugation, the gradient solution is passed through an absorbance recorder to generate a polysome profile. Ten to twelve fractions (0.8-1 mL each) are collected for isolating different RNA and protein populations. The overall method is tedious and lengthy (6-9 h), requires access to a suitable ultracentrifuge rotor and centrifuge, and requires a substantial amount of tissue material, which can be a limiting factor. Moreover, there is often a dilemma over the quality of RNA and protein populations in the individual fractions due to the extended experiment times. To overcome these challenges, here we describe a miniature sucrose gradient for polysome profiling using Arabidopsis thaliana seedlings that takes ~1 h centrifugation time in a tabletop ultracentrifuge, reduced gradient synthesis time, and also less tissue material. The protocol described here can be easily adapted to a wide variety of organisms and polysome profiling of organelles, such as chloroplasts and mitochondria. Key Features • Mini sucrose gradient for polysome profiling that requires less than half the processing time vs. traditional methods. • Reduced starting tissue material and sample volume for sucrose gradients. • Feasibility of RNA and protein isolation from polysome fractions. • Protocol can be easily modified to a wide variety of organisms (and even polysome profiling of organelles, such as chloroplast and mitochondria). Graphical Overview.