Iodixanol gradient ultracentrifugation

With the cytoplasmic fractions, we performed isopycnic iodixanol gradient subcellular fractionation as previously described ²³. To prepare a 50% (w/v) iodixanol working solution, we added five volumes of OptiPrep (Cosmo Bio USA, Carlsbad, CA, USA) to 1 vol of 0.25 M sucrose, 6 mM EDTA, 60 mM HEPES, pH 7.4. OptiPrep™ is a 60% (w/v) solution of iodixanol in water, density = 1.32 g/ml. Following the steps previously described ²⁴ and associated protocols on the Axis-Shield website (http://www.axis-shield-density-gradient-media.com/), we mixed a certain ratio of homogenization medium (a HEPES based buffer [10 mM, pH 7.4] with 0.25 M sucrose, 1 mM EDTA, and 2 mM ATP) with certain volumes of iodixanol working solution to prepare the 2.5 ml gradient dilutions of 8%, 16%, 28%, and 38% each of iodixanol (supplement protocol 1). The osmolality of these dilutions is in the range of 295-310 mOsm. We layered 2.5 ml of each dilution into Beckman Ultra-Clear 13.2ml centrifuge tubes. Cytoplasmic lysates were carefully loaded onto the discontinuous gradient and, using an SW41Ti rotor and a Beckman L8-55 ultracentrifuge, the samples were centrifuged at 28,500 rpm (100 000 g) for 18 hours at 4 °C. Eighteen to twenty fractions (approximately 500 µl) were collected using a Labconco Auto Densi-Flow peristaltic pump and stored at -20 °C (Fig. ​(Fig.1A).1A). Fraction densities were determined by weighing a known volume using an analytical balance.

Experimental design of proteasome study. (A) Overview of the experimental study. Cytoplasmic and nuclear lysates were subjected to an iodixanol gradient fractionation. Twenty fractions were collected and analyzed for proteasomal catalytic activities with and without proteasome inhibitors. (B) Structure of the 20S, 26S, and 30S proteasomes. The proteasome can be composed of three particles: one is the catalytic core or 20S particle (yellow and orange); for the other two, the 19S regulatory particle consists of 19 individual proteins divided into a 10-protein 'base' (blue) subassembly and a 9-protein 'lid' sub-particle (green), generating either 26S (one 19S cap) or 30S (two 19S caps). (C) Graphs show the linear correlation between fluorescence intensity and the amount of substrate-AMC-hydrolyzing activity in reaction, plotted as arbitrary fluorescence units (AFU). The fluorescence intensity was measured at 380 nm excitation and 460 nm minus background fluorescence.