Microsomal membrane isolation

Harvest cells by detachment with phosphate buffered saline (PBS, Sigma, D8537-500ml) + 0.2% EDTA.

Pellet cells by centrifugation (5 min, 400 g_avg, 4°C).

Wash cell pellet in phosphate buffered saline (PBS, Sigma, D8537-500ml). Repeat 2 times.

Pellet cells by centrifugation (5 min, 400 g_avg, 4°C).

Resuspend pellet in hypnotic LIS buffer (10 mM Tris HCl pH 7.5, 0.5 mM MgCl₂, 1 mM DTT) supplemented with protease inhibitor (Sigmafast, Sigma, S8830-20TAB).

Incubate 15 min on ice.

Transfer the suspension to a dounce homogenizer and complete 60 up-and-down strokes.

Add an equal volume of 1 M buffer (0.5 M sucrose, 10 mM Tris HCl, pH 7.5, 40 µM CaCl₂, 1 mM DTT) supplemented with protease inhibitor.

Complete another 30 up-and-down strokes.

Centrifuge lysate (10min, 1000 g_avg, 4°C) to pellet and remove nuclear fraction.

Collect supernatant to continue isolation.

Centrifuge supernatant from previous step (20min, 15 000 g_avg, 4°C) to pellet the mitochondrial/lysosomal fraction.

Collect supernatant to continue isolation (pelleted mitochondrial/lysosomal fraction can be stored for future analysis).

Centrifuge supernatant from previous step (35 min, 140 000 g_avg, 4°C) to pellet the microsomal fraction.

Resuspend pellets in 0.25 M sucrose, 1 mM DTT and supplemented with protease inhibitors.

Snap freeze the resuspended mitochondrial/lysosomal and microsomal fractions in liquid nitrogen.

Store at -80 °C.