Subcellular fractionation by discontinuous sucrose density gradient centrifugation

Three T-75 flasks grown to confluency were placed on ice, washed once with ice-cold PBS (pH 7.4) to remove growth medium, and followed by incubation in ice-cold 10 mM tris-HCl (pH 7.4) for 1 min to induce osmotic swelling as an aid to cell disruption. The buffer was rapidly removed, and cells were scraped into an ice-cold homogenization HES buffer composed of 20 mM Hepes (pH 7.4), 1 mM EDTA, 0.25 M sucrose, and protease inhibitors. Cells were mechanically lysed in the absence of detergent by passing them repeatedly through 25G1/2 and 26G1/2 needles. The procedure for subcellular fractionation was adapted from a previous protocol (50). Briefly, cell lysates were centrifuged for 10 min at 1000g at 4°C in a benchtop centrifuge to obtain a p1000 pellet containing cell nuclei and unbroken cells. The p1000 pellet was resuspended in 1 ml of homogenization buffer, sonicated 3× for 10 s each using a Transonic digital sonicator at an amplitude setting of 100, and stored as p1000. The post-p1000 supernatant was then centrifuged for 10 min at 8000g at 4°C to obtain a p8000 pellet containing PM, mitochondria, and ER. The p8000 pellet was resuspended in 1 ml of homogenization buffer, sonicated, and then stored as p8000. The post-8000g supernatant was loaded at the top of a continuous 10 to 40% (w/v) sucrose density gradient prepared using a 15-ml Gradient Mixer (Z340391, Sigma-Aldrich), subjected to ultracentrifugation for 18 hours at 4°C at 35,000 rpm using a swing SW41 Beckman rotor. Then, 1-ml gradient fractions were harvested beginning from the top of the tube and stored at −80°C.

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